Genes for male accessory gland proteins in Drosophila melanogaster

ABSTRACT

The present invention provides a number of accessory gland proteins from  Drosophila . The invention also provides an accessory gland protein which is toxic to insect cells and can be used to kill or inhibit the development of insects. Methods of controlling insects are also provided.

The present application is a divisional of U.S. patent application Ser. No. 10/114,774, filed Apr. 4, 2002, which is a divisional of U.S. patent application Ser. No. 09/219,983, filed Dec. 23, 1998, now U.S. Pat. No. 6,380,159, issued Apr. 30, 2002, which claims the benefit of U.S. Provisional Patent Application Ser. No. 60/071,315, filed Dec. 23, 1997.

The subject matter of this application was made with support from the United States Government under Grant Nos. IBN97-23356, IBN94-06171, and DCB91-08221 from the National Science Foundation. The United States Government may retain certain rights.

BACKGROUND OF THE INVENTION

After mating, behavioral and physiological changes are seen in the female insect. Compared to virgins, mated Drosophila melanogaster females are largely unreceptive to further mating, lay eggs at an elevated rate, live less long, and store and efficiently utilize sperm (reviewed in Hall, 1994; Chen, 1996; Kubli, 1996; Wolfner, 1997). These changes in the female occur because she receives, via seminal fluid, secretions from her mate's accessory gland and also sperm (see Chen, 1996; Kubli, 1996; Wolfner, 1997 for reviews and for original references). Products of the predominant cell type of the accessory gland, the main cells, are necessary for changes in the female's egg-laying rate and receptivity on the first day after mating. Stored sperm cause these effects to persist for up to 11 days following mating. Accessory gland main cell secretions also shorten the life span of the mated female. In addition, they play a role in the storage of sperm and in the competition between sperm from sequential matings.

Knowledge of how accessory gland products mediate these changes is important in understanding the control of insect fertility and the mechanisms of peptide hormone action. Once genes encoding Accessory gland proteins (Acps) are identified, genetic and molecular genetic techniques uniquely possible in Drosophila can be used to dissect the role of each protein in reproduction. In a few cases, it is possible to identify the functions of Acps by injecting purified fractions into unmated female flies and observing behavioral effects. For example, in D. melanogaster, a “sex peptide” (“SP”) of 36 amino acids was purified and shown to stimulate egg-laying and depress receptivity to mating for one day (Chen, 1988). SP was cloned and shown to derive from a single gene at chromosomal position 70A (Chen, 1988). A sex peptide and a second peptide, ovulation-stimulating substance (OSS), with similar activities have also been purified from D. suzukii (Ohashi, 1991; Schmidt, 1993).

Only Acps which can be purified or synthesized in active forms, act on their own and act via the hemolymph, can be identified by such assays. In order to identify Acp genes without presupposition of function, differential cDNA hybridization can be used to isolate RNAs expressed only in accessory glands (Schäfer, 1986; DiBenedetto, 1987; Monsma and Wolfner, 1988). cDNA hybridization screens are more likely to isolate abundant RNAs in a tissue. Thus, they are biased towards RNAs expressed in main cells of the accessory gland (96% of the secretory cells of the accessory gland; Bertram, 1992) rather than the rarer secondary cells (4% of the secretory cells of the gland; Bertram, 1992). Previous differential cDNA hybridization screens for genomic clones encoding male-specific transcripts identified three genomic regions encoding Acps (Schäfer, 1986; DiBenedetto, 1987). Of these, the 95EF region encodes a small secreted Acp (DiBenedetto, 1990), 57D contains a gene cluster encoding three small peptides (Simmerl, 1995), and the 51F locus has not yet been characterized. In addition to these genes, a region encoding two Acps has been identified by screening a “chromosomal walk” for accessory gland-specific transcription units (Monsma and Wolfner, 1988). In this region, only 20 bases separate the gene for Acp26Aa, an ELH-similar prohormone-like molecule (Monsma and Wolfner, 1988) that stimulates egg-laying in the mated female fly (Herndon and Wolfner, 1995), from the gene for Acp26Ab, a small peptide of as yet unknown function (Monsma and Wolfner, 1988). The previously-isolated Acp genes are only a small subset of Acp genes, though the total number of Acp genes is difficult to estimate from prior protein electrophoretic data (e.g. Ingman-Baker and Candido, 1980; Stumm-Zollinger and Chen, 1985; Whalen and Wilson, 1986; Coulthart and Singh, 1988) as summarized and discussed in Chen (1991). This is because on the one hand in the electrophoretic studies small peptides were not resolved, while on the other hand some Acps run as multiple bands on SDS gels (Monsma and Wolfner, 1988). To gain a more complete picture of the spectrum of proteins produced by the accessory gland, a differential screen aimed directly at accessory gland-specific RNAs was performed.

SUMMARY OF THE INVENTION

The present invention provides an isolated nucleic acid molecule encoding an accessory gland protein from Drosophila which has the biological property of an insect toxin.

The invention further provides an isolated nucleic acid molecule encoding an amino acid sequence sufficiently duplicative of the accessory gland protein encoded by the nucleic acid molecule of SEQ ID NO:2 so that a polypeptide expressed from the nucleic acid molecule has the biological property of an insect toxin.

Another embodiment of the invention is an isolated Drosophila melanogaster insect toxin protein.

The invention also provides a method of reducing an insect's life span. The insect is contacted with an isolated Drosophila insect toxin protein under conditions effective shorten the insect's life span.

Yet another embodiment of the invention is a method of reducing an insect's life span by contacting the insect with an expression vector containing a nucleic acid molecule encoding a Drosophila insect toxin protein under conditions effective to express the protein.

The invention also provides isolated nucleic acid molecules having the nucleotide sequences of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:13, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:27, or SEQ ID NO:30, or a nucleic acid molecules which hybridizes under stringent conditions to a nucleic acid molecule having a nucleic acid sequence of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:13, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:27, or SEQ ID NO:30.

The present invention also provides isolated proteins having an amino acid sequence of SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO: 12, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, or SEQ ID NO:32.

Yet another embodiment of the invention is a method for determining whether a female Drosophila melanogaster has recently mated. An antibody, fragment thereof, or probe which recognizes a protein having a sequence provided in SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, or SEQ ID NO:32 is provided. The antibody, fragment thereof, or probe is bound to a label effective to permit detection of the protein upon binding of the antibody, fragment thereof, or probe to the protein. The labeled antibody is contacted with a fluid or tissue sample from Drosophila melanogaster under conditions effective to permit binding of the antibody, fragment thereof, or probe to the protein. The presence of any of the protein in the biological sample is detected by detecting the label.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the genomic organization and transcript characteristics of new Acp genes. Gene name, position and copy number: The genes are named Acp followed by their location on the polytene chromosomes as determined by in situ hybridization. The site for Acp76A is determined as 75F/76A, for 63F is 63F/64A, for 31F is 31F/32A. The genomic clone containing Acp33A has a minor site of hybridization at 32E. Restriction map: The map of the genomic region containing the accessory gland transcript is shown, to a scale such that the leftmost EcoRI-XbaI fragment of Acp31F is 1.0 kb. In all cases but 2, the map is derived from a single genomic clone of the region. The larger region covered for Acps 36DE and 33A is a composite of the overlapping maps of three genomic clones each. The genomic fragment(s) that hybridize to the Acp transcript is shown as a thicker line. The clones are oriented with the Acp gene transcription direction from left to right, except for Acps 31F, 53Eb and rep1, where the transcription direction was not determined. E=Eco RI, H=Hin dIII, B=Bam HI, X=Xba I, S=Sal I, Bgl=Bgl II, N=Ngo M1, P=Pml I, W=Swa I. Transcript characteristics: The size of each poly A⁺ accessory gland transcript was determined on a minimum of three independent Northern blots. The number of bases (nt) in the cDNA clone before the first AUG is listed (5′ UTR); since the cDNA clones may not be full-length, the size of this untranslated leader should be taken as a minimum. For all genes except Acp36DE, the longest open reading frame begins with the first AUG. As noted in the text, although Acp36DE has a short open reading frame beginning at base 18 of the cDNA sequence (and preceded by AUUA), the Acp is encoded by the longer second open reading frame which is the one listed in the figure.

The four bases immediately 5′ of the initiating AUG are shown; those matching consensus (C/A A A C/A; Cavener, 1987) are underlined. The column headed 3′UTR gives the number of bases from the translational stop codon to the beginning of the poly A tail. For Acp53Ea, the cDNA clone did not contain the polyA tail. Thus the size of its UTR is a minimum. The distance of that tail from the AAUAAA signal (or its best, 5/6, match) is given in the “pA sig.” column.

The GenBank accession numbers for these sequences are given. For Acps 32CD and 98AB, the sequences reported are composites of overlapping genomic and cDNA sequences. GenBank requires that genomic and cDNA sequences be given separate accession numbers. For each of these genes, the first number listed is for the genomic sequence, the second for its partially-overlapping cDNA. For Acp76A, the accession number is for genomic sequence, which completely contains the cDNA sequence of this apparently intron-less mRNA. The other accession numbers are for cDNA sequences. Two accession numbers are given for Acp33A, since GenBank requires that each predicted ORF in this single mRNA be listed with its own accession number. The nucleotide sequences for these two accession numbers are, of course, the same. Characteristics of predicted Acp: The number of amino acids (aa) in the Acp-encoding ORF, the length of the hydrophobic sequence terminating at a predicted signal sequence cleavage site (von Heijne, 1983) and predicted features of the Acp are listed in successive columns.

FIGS. 2A and 2B show the expression of an accessory gland gene, Acp36DE, as an example. FIG. 2A is a representative Northern blot of male accessory gland RNA and female RNA probed with radiolabeled RNA complementary to Acp36DE. A male specific RNA of 2.6-2.7 kb is detected. The blot was probed separately with sequences complementary to actin as a loading control as in DiBenedetto (1987), which confirmed that equivalent amounts of RNA were present in the two lanes. FIG. 2B provides a representative in situ hybridization to whole-mount accessory glands from a 3-day-old virgin male. The probe was the digoxigenin-labeled 2.1 kb EcoRI fragment of Acp36DE cDNA clone #11A (Bertram, 1994). Staining is seen in the accessory glands (“ag”), but not in the ejaculatory duct (“ed”). Bar=0.128 millimeter. FIG. 2B (inset) is a higher magnification of the distal tip of an accessory gland from a similar in situ hybridization. Main cells are stained. Secondary cells are not stained. This can be seen most clearly at the edge of the gland, where an arrowhead points to an unstained secondary cell (the apparent “bite” taken out of the edge of the stained tissue is the unstained cell). Other secondary cells, lying atop a layer of stained main cells and themselves surrounded by stained main cells, are seen as light circles. Bar=0.051 millimeter.

FIG. 3 provides the predicted protein sequences of new Acps. The predicted protein sequence from the single long ORF in each Acp's cDNA is shown. For Acp33A, both ORFs are shown; ORF1 is the more 5′ one. The potential signal sequence is written in lower case. Potential N-linked glycosylation sites (N-x-S/T/C; Kornfeld and Kornfeld, 1985; Miletich and Broze, 1990), amidation site (IGKK; Kreil, 1984; Bradbury and Smyth, 1987), glycosaminoglycan attachment sites (SGxG; Hassell, 1986; Bourdon, 1987) and basic amino acids (K, R) in contexts consistent with prohormone processing cleavages (Schwartz, 1986; Benoit, 1987; Nakayama, 1992) are underlined. The amino acids that match serpin consensus in Acp76A, and those in Acp62F that are similar to toxin PhTx2-6 are boxed. Sequencing of genomic DNA upstream from the 5′ end of the incomplete cDNA of Acp32CD led to the discovery of an ORF encoding 329 amino acids. This ORF is too long to be encoded on the 0.95 kb Acp32CD RNA, whose size includes a poly A tail. Therefore, it is surmised that Acp32CD corresponds to the 241 amino acid sequence shown in the figure, which begins at the second AUG of the 329-amino acid ORF (AGAU immediately precedes the AUG of the long ORF). The amino acids encoded by genomic sequence immediately upstream of the sequence shown in this figure are: mppllrhcfg hafiglplfn GQEQPRPQSN RFDSGQRRSS LYIRDGRTAR AAQRCSDVAD ADAATHWLLG PVALGQLPEH GALGQKYY (SEQ ID NO:1).

FIG. 4 is a best-fit alignment of similar regions of Acp62F (aa residues 46-73 of SEQ ID NO:4) and PhTx2-6 (SEQ ID NO:35, aa residues 9-35 of the full length PhTx2-6 protein).

FIG. 5 shows the rate of survival of baculovirus injected 5^(th) instar Trichoplusia ni. The results of controls and baculovirus which express Acp62F are included.

FIG. 6 shows the rate of pupation of 3^(rd) instar Trichoplusia ni after injection with Acp62F protein.

FIG. 7 shows the effect on survival of Acp62F expression in pre-adult fruit flies.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides isolated genes encoding 12 previously unreported accessory gland-specific mRNAs from the fruit fly Drosophila melanogaster. The restriction maps of the new genes, the chromosome positions—which are all autosomal—of the 11 non-repetitive genes, their expression patterns, and the sequences of the Accessory gland proteins (Acps) encoded by 9 of the genes are provided.

The present invention provides an isolated nucleic acid molecule encoding an accessory gland protein from Drosophila which has the biological property of an insect toxin. The 115 amino acid Acp62F has a 28 amino acid region of high sequence similarity to a neurotoxin of the Brazilian armed spider Phoneutria nigriventer. In a preferred embodiment of the invention, the accessory gland protein has greater than 40% sequence similarity to amino acids 9 to 35 of the PhTx2-6 toxin of Phoneutria nigriventer. This protein has been tested and has been shown to kill or inhibit the growth of insects. In particular, the accessory gland protein is toxic to Drosophila or trichoplusia.

The present invention provides a gene encoding Acp62F. The cDNA sequence of the Acp62F gene is as follows (SEQ ID NO:2): 1 ggtagacgta ttccccatct acaatgacgg acatgtggag cttgaagatc tgtgcctgtc 61 tgggccttct attacttttc aaacccatcg actccatggg atggcaagga cctaaagttg 121 actgtacggc caacggaact cagacggagt gtcctgtagc atgtcctgaa acctgcgagt 181 actccggcaa tggaccctgc gtcaagatgt gcggagctcc ttgtgtgtgt aagccgggat 241 atgttatcaa tgagaggatt ccggcctgtg ttctgcgatc cgattgccca aaagatgttg 301 ttcgaaagga agatatgcta ctgggtgtat cgaactttaa gtgctttagc agaaattaca 361 actgttcata gaaatttatt aggaaggcag ctaaacttta aactaaaata caataaaatg 421 taaataaaaa aaaaaaaaa

In a preferred embodiment of the invention, the isolated nucleic acid moleucle has a nucleotide sequence according to SEQ ID NO:2, or a nucleic acid molecule which hybridizes under stringent conditions to a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO:2.

The nucleic acid molecule can be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), genomic or recombinant, biologically isolated or synthetic. The invention encompasses the DNA sequences as well as their complements. The DNA molecule can be a cDNA molecule, which is a DNA copy of a messenger RNA (mRNA) encoding the accessory gland protein. A suitable RNA molecule is mRNA.

Suitable nucleic acid molecules include those nucleic acid molecules encoding an accessory gland protein and having a nucleotide sequence which is at least 95% homologous to the nucleotide sequence of an accessory gland protein of the present invention.

While the nucleotide sequence is at least 95% homologous, nucleotide identity is not required. As should be readily apparent to those skilled in the art, various nucleotide substitutions are possible which are silent mutations (i.e. the amino acid encoded by the particular codon does not change). It is also possible to substitute a nucleotide which alters the amino acid encoded by a particular codon, where the amino acid substituted is a conservative substitution (i.e. amino acid “homology” is conserved).

Alternatively, suitable DNA sequences may be identified by hybridization to the disclosed sequences which encode the accessory gland proteins under stringent conditions. For example, sequences can be isolated that hybridize to a DNA molecule comprising a nucleotide sequence of approximately 50 continuous bases of the sequences encoding the accessory gland proteins under stringent conditions characterized by a hybridization buffer comprising 0.9M sodium citrate (“SSC”) buffer at a temperature of 37° C. and remaining bound when subject to washing with the SSC buffer at 37° C.; and preferably in a hybridization buffer comprising 20% formamide in 0.9M saline/0.09M SSC buffer at a temperature of 42° C. and remaining bound when subject to washing at 42° C. with 0.2× SSC buffer at 42° C.

Another embodiment of the invention is an expression vector carrrying the nucleic acid molecule encoding Acp62F.

The DNA molecule encoding the accessory gland proteins can be incorporated in cells using conventional recombinant DNA technology. Generally, this involves inserting the DNA molecule into an expression system to which the DNA molecule is heterologous (i.e. not normally present). The heterologous DNA molecule is inserted into the expression system or vector in proper sense orientation and correct reading frame. The vector contains the necessary elements for the transcription and translation of the inserted protein-coding sequences.

U.S. Pat. No. 4,237,224 to Cohen and Boyer, which is hereby incorporated by reference, describes the production of expression systems in the form of recombinant plasmids using restriction enzyme cleavage and ligation with DNA ligase. These recombinant plasmids are then introduced by means of transformation and replicated in unicellular cultures including procaryotic organisms and eucaryotic cells grown in tissue culture.

Recombinant genes may also be introduced into viruses, such as vaccina virus. Recombinant viruses can be generated by transfection of plasmids into cells infected with virus.

Suitable vectors include, but are not limited to, the following viral vectors such as lambda vector system gt11, gt WES.tB, Charon 4, and plasmid vectors such as pBR322, pBR325, pACYC177, pACYC1084, pUC8, pUC9, pUC18, pUC19, pLG339, pR290, pKC37, pKC101, SV 40, pBluescript II SK +/− or KS +/− (see Stratagene, 1993), pQE, pIH821, pGEX, pET series (see Studier, 1990), and any derivatives thereof. Recombinant molecules can be introduced into cells via transformation, particularly transduction, conjugation, mobilization, or electroporation. The DNA sequences are cloned into the vector using standard cloning procedures in the art, as described by Sambrook, (1989).

A variety of host-vector systems may be utilized to express the protein-encoding sequence(s). Primarily, the vector system must be compatible with the host cell used. Host-vector systems include but are not limited to the following: bacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA; microorganisms such as yeast containing yeast vectors; mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g., baculovirus); and plant cells infected by bacteria. The expression elements of these vectors vary in their strength and specificities. Depending upon the host-vector system utilized, any one of a number of suitable transcription and translation elements can be used.

In a preferred embodiment, the expression vector is a viral vector. In a more preferred embodiment, the viral vector is a baculovirus vector. Baculovirus vectors, which are derived from the genome of the AcNPV virus, are designed to provide high level expression of cDNA in the Sf9 line of insect cells. A baculovirus vector is constructed in accordance with techniques that are known in the art, for example, as described in Kitts, (1993); Smith, (1983); and Luckow and Summer, (1989). In one embodiment of the present invention, a baculovirus expression vector is constructed substantially in accordance to Summers and Smith, (1987). Moreover, materials and methods for baculovirus/insect cell expression systems are commercially available in kit form, for example, the MaxBac Registered TM kit from Invitrogen (San Diego, Calif.). Recombinant baculoviruses are generated by homologous recombination following co-transfection of the baculovirus transfer vector and linearized AcNPV genomic DNA (Kitts, 1990) into Sf9 cells.

In yet another embodiment, the invention provides a host cell having the expression vector carrying the accessory gland protein gene. In a preferred embodiment, the host cell is an insect cell.

Different genetic signals and processing events control many levels of gene expression (e.g., DNA transcription and messenger RNA (mRNA) translation).

Transcription of DNA is dependent upon the presence of a promoter which is a DNA sequence that directs the binding of RNA polymerase and thereby promotes mRNA synthesis. The DNA sequences of eucaryotic promoters differ from those of procaryotic promoters. Furthermore, eucaryotic promoters and accompanying genetic signals may not be recognized in or may not function in a procaryotic system, and, further, procaryotic promoters are not recognized and do not function in eucaryotic cells.

Similarly, translation of mRNA in procaryotes depends upon the presence of the proper procaryotic signals which differ from those of eucaryotes. Efficient translation of mRNA in procaryotes requires a ribosome binding site called the Shine-Dalgarno (“SD”) sequence on the mRNA. This sequence is a short nucleotide sequence of mRNA that is located before the start codon, usually AUG, which encodes the amino-terminal methionine of the protein. The SD sequences are complementary to the 3′-end of the 16S rRNA (ribosomal RNA) and probably promote binding of mRNA to ribosomes by duplexing with the rRNA to allow correct positioning of the ribosome. For a review on maximizing gene expression, see Roberts and Lauer, (1979).

Promoters vary in their “strength” (i.e. their ability to promote transcription). For the purposes of expressing a cloned gene, it is desirable to use strong promoters in order to obtain a high level of transcription and, hence, expression of the gene. Depending upon the host cell system utilized, any one of a number of suitable promoters may be used. For instance, when cloning in E. coli, its bacteriophages, or plasmids, promoters such as the T7 phage promoter, lac promoter, trp promoter, recA promoter, ribosomal RNA promoter, the PR and PL promoters of coliphage lambda and others, including but not limited, to lacUV5, ompF, bla, lpp, and the like, may be used to direct high levels of transcription of adjacent DNA segments. Additionally, a hybrid trp-lacUV5 (tac) promoter or other E. coli promoters produced by recombinant DNA or other synthetic DNA techniques may be used to provide for transcription of the inserted gene.

Bacterial host cell strains and expression vectors may be chosen which inhibit the action of the promoter unless specifically induced. In certain operations, the addition of specific inducers is necessary for efficient transcription of the inserted DNA. For example, the lac operon is induced by the addition of lactose or IPTG (isopropylthio-beta-D-galactoside). A variety of other operons, such as trp, pro, etc., are under different controls.

Specific initiation signals are also required for efficient gene transcription and translation in procaryotic cells. These transcription and translation initiation signals may vary in “strength” as measured by the quantity of gene specific messenger RNA and protein synthesized, respectively. The DNA expression vector, which contains a promoter, may also contain any combination of various “strong” transcription and/or translation initiation signals. For instance, efficient translation in E. coli requires an SD sequence about 7-9 bases 5′ to the initiation codon (“ATG”) to provide a ribosome binding site. Thus, any SD-ATG combination that can be utilized by host cell ribosomes may be employed. Such combinations include but are not limited to the SD-ATG combination from the cro gene or the N gene of coliphage lambda, or from the E. coli tryptophan E, D, C, B or A genes. Additionally, any SD-ATG combination produced by recombinant DNA or other techniques involving incorporation of synthetic nucleotides may be used.

Once the isolated DNA molecule encoding the accessory gland protein has been cloned into an expression system, it is ready to be incorporated into a host cell. Such incorporation can be carried out by the various forms of transformation noted above, depending upon the vector/host cell system. Suitable host cells include, but are not limited to, bacteria, virus, yeast, mammalian cells, insect, plant, and the like.

The invention also provides an antisense nucleic acid molecule that is complementary to the mRNA encoding the the accessory gland protein, or a fragment thereof capable of hybridizing under stringent conditions to the mRNA. The antisense nucleic acid molecule is ribonucleic acid. This antisense molecule can base-pair with the mRNA, preventing translation of the mRNA into protein.

The invention further provides an isolated fragment of the nucleic acid molecule encoding the accessory gland protein. Nucleic acid molecules encoding the accessory gland proteins, and fragments of the nucleic acid molecules, are thus provided.

Each of the nucleic acid molecules, fragments thereof, antisense nucleic acid molecules, and fragments thereof, can be expressed in suitable host cells using conventional techniques. Such techniques may involve the use of expression vectors which comprise the nucleic acid molecules, fragments thereof, antisense nucleic acid molecules, or fragments thereof. These expression vectors can then be used to transform suitable host cells.

Host cells transformed with nucleic acid molecules encoding the accessory gland protein can be used to produce accessory gland proteins (or cells transformed with the fragments can be used to produce fragments of the accessory gland proteins). Alternatively, the fragments or full-length accessory gland proteins can be produced synthetically using the sequence information of the accessory gland proteins and fragments. In host cells transformed with the antisense nucleic acid molecules, or fragments thereof, the antisense nucleic acid molecules or fragments thereof will block translation of the accessory gland protein. Accordingly, in host cells transformed with the antisense nucleic acid molecules or fragments thereof, the expression of accessory gland protein is decreased.

The invention also provides an isolated nucleic acid molecule having a nucleic acid sequence encoding an amino acid sequence sufficiently duplicative of the accessory gland protein encoded by the disclosed nucleic acid molecules so that a polypeptide expressed from the nucleic acid molecule has the biological property of an insect toxin. The cDNA for Acp62F includes an open reading frame beginning at position 24 of the cDNA sequence. The open reading frame sequence is provided in SEQ ID NO:3, as follows: 1 atgacggaca tgtggagctt gaagatctgt gcctgtctgg gccttctatt acttttcaaa 61 cccatcgact ccatgggatg gcaaggacct aaagttgact gtacggccaa cggaactcag 121 acggagtgtc ctgtagcatg tcctgaaacc tgcgagtact ccggcaatgg accctgcgtc 181 aagatgtgcg gagctccttg tgtgtgtaag ccgggatatg ttatcaatga gaggattccg 241 gcctgtgttc tgcgatccga ttgcccaaaa gatgttgttc gaaaggaaga tatgctactg 301 ggtgtatcga actttaagtg ctttagcaga aattacaact gttca

The present invention also provides an isolated Drosophila melanogaster insect toxin protein. The amino acid sequence of the Acp62F protein is provided in SEQ ID NO:4, as follows: 1 MTDMWSLKIC ACLGLLLLFK PIDSMGWQGP KVDCTANGTQ TECPVACPET CEYSGNGPCV 61 KMCGAPCVCK PGYVINERIP ACVLRSDCPK DVVRKEDMLL GVSNFKCFSR NYNCS

In a preferred embodiment, the isolated Drosophila melanogaster insect toxin protein has greater than 40% sequence similarity to amino acids 9 to 35 of the PhTx2-6 toxin of Phoneutria nigriventer. Another preferred embodiment is where the isolated Drosophila melanogaster insect toxin protein is toxic to Drosophila or caterpillar.

The isolated Drosophila melanogaster insect toxin protein may have an amino acid sequence according to SEQ ID NO:4, or be a protein encoded by a nucleic acid molecule which hybridizes under stringent conditions to a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO:2 and which has the biological property of an insect toxin.

The protein or polypeptide of the present invention is preferably produced in purified form (preferably at least about 80%, more preferably 90%, pure) by conventional techniques. In accordance with the present invention, the accessory gland proteins are isolated from host cells. Methods for protein isolation are known in the art. Generally, proteins can be purified by conventional chromatography, including gel-filtration, ion-exchange, and immunoaffinity chromatography, by high-performance liquid chromatography, such as reversed-phase high-performance liquid chromatography, ion-exchange high-performance liquid chromatography, size-exclusion high-performance liquid chromatography, high-performance chromatofocusing and hydrophobic interaction chromatography, etc., by electrophoretic separation, such as one-dimensional gel electrophoresis, two-dimensional gel electrophoresis, etc. Such methods are known in the art. See for example Ausubel (1994). Additionally, antibodies can be prepared against substantially pure preparations of the protein. See, for example, Radka, (1983) and Radka, (1984). Any combination of methods may be utilized to purify protein having pesticidal properties. As the protocol is being formulated, pesticidal activity is determined after each purification step, if relevant.

Fragments of the above polypeptide or protein are also encompassed by the present invention. Suitable fragments can be produced by several means. In the first, subclones of the gene encoding the protein of the present invention are produced by conventional molecular genetic manipulation by subcloning gene fragments. The subclones then are expressed in vitro or in vivo in bacterial cells to yield a smaller protein or peptide that can be tested for an accessory gland protein.

As an alternative, fragments of an accessory gland protein can be produced by digestion of an accessory gland protein with proteolytic enzymes like chymotrypsin or Staphylococcus proteinase A, or trypsin. Different proteolytic enzymes are likely to cleave accessory gland proteins at different sites based on the amino acid sequence of an accessory gland protein.

In another approach, based on knowledge of the primary structure of the protein, fragments of the accessory gland protein gene may be synthesized by using the PCR technique together with specific sets of primers chosen to represent particular portions of the protein. These then would be cloned into an appropriate vector for increased expression of an accessory peptide or protein.

Chemical synthesis can also be used to make suitable fragments. Such a synthesis is carried out using known amino acid sequences for the accessory gland protein being produced. Alternatively, subjecting a full length accessory gland protein to high temperatures and pressures will produce fragments. These fragments can then be separated by conventional procedures (e.g., chromatography, SDS-PAGE).

Variants may also (or alternatively) be modified by, for example, the deletion or addition of amino acids that have minimal influence on the properties, secondary structure, and hydropathic nature of the polypeptide. For example, a polypeptide may be conjugated to a signal (or leader) sequence at the N-terminal end of the protein which co-translationally or post-translationally directs transfer of the protein. The polypeptide may also be conjugated to a linker or other sequence for ease of synthesis, purification, or identification of the polypeptide.

Gene amplification can also be used to obtain very high levels of expression of transfected gene. When cell cultures are treated with methotrexate (“Mtx”), an inhibitor of a critical metabolic enzyme, dihydrofolate reductase (“DHFR”), most cells die, but eventually some Mtx-resistant cells grow up. A gene to be expressed in cells is cotransfected with a cloned DHFR gene, and the transfected cells are subjected to selection with a low concentration of Mtx. Resistant cells that have taken up the DHFR gene (and, in most cases, the cotransfected gene) multiply. Increasing the concentration of Mtx in the growth medium in small steps generates populations of cells that have progressively amplified the DHFR gene, together with linked DNA. Although this process takes several months, the resulting cell cultures capable of growing in the highest Mtx concentrations will have stably amplified the DNA encompassing the DHFR gene a hundredfold or more, leading to significant elevation of the expression of the cotransfected gene.

Once the nucleic acid molecule encoding an accessory gland protein has been inserted into a host cell, with or without the use of an intermediate expression vector, the host cell can be used to produce the accessory gland protein by culturing the cell under conditions suitable for translation of the DNA molecule, thereby expressing the accessory gland protein. The accessory gland protein can then be recovered from the cell. Generally, the accessory gland protein of the present invention is produced in purified form by conventional techniques, such as by secretion into the growth medium of recombinant E. coli. To isolate the protein, the E. coli host cell carrying a recombinant plasmid is propagated, homogenized, and the homogenate is centrifuged to remove bacterial debris. The supernatant is then subjected to sequential ammonium sulfate precipitation. The fraction containing the protein of the present invention is subjected to gel filtration in an appropriately sized dextran or polyacrylamide column to separate the proteins. If necessary, the protein fraction may be further purified by HPLC.

The present invention also provides an isolated antibody, fragment thereof, or probe which recognizes an accessory gland protein provided by the invention. Preferred antibodies, fragments thereof, or probes are those which recognize a protein having an amino acid sequence according to SEQ ID NO:4, or a protein encoded by a nucleic acid molecule which hybridizes under stringent conditions to a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO:2 and where the protein has the biological function of an insect toxin.

Antibodies can also be raised to each of the accessory gland proteins, and to the isolated fragments thereof. Antibodies of the subject invention include polyclonal antibodies and monoclonal antibodies which are specific for an accessory gland protein or isolated fragments thereof. In addition to utilizing whole antibodies, the present invention encompasses use of binding portions of such antibodies. Such binding portions include Fab fragments, F(ab′)2 fragments, and Fv fragments. Such antibody fragments can be made by conventional procedures, such as proteolytic fragmentation procedures, as described in Goding, (1983). These antibodies or fragments thereof can thus be used to detect the presence of an accessory gland protein in a sample (or to detect the presence of a fragment of an accessory gland protein), by contacting the sample with the antibody or fragment thereof. The antibody or fragment thereof binds to an accessory gland protein or fragment thereof present in the sample, forming a complex therewith. The complex can then be detected, thereby detecting the presence of the accessory gland protein or fragment thereof in the sample.

In a preferred embodiment the antibody, fragment thereof, or probe is a monoclonal antibody. Alternatively, the antibody, fragment thereof, or probe is a polyclonal antibody.

Monoclonal antibody production may be effected by techniques which are well-known in the art. Basically, the process involves first obtaining immune cells (lymphocytes) from the spleen of a mammal (e.g., mouse) which has been previously immunized with the antigen of interest either in vivo or in vitro. The antibody-secreting lymphocytes are then fused with (mouse) myeloma cells or transformed cells, which are capable of replicating indefinitely in cell culture, thereby producing an immortal, immunoglobulin-secreting cell line. The resulting fused cells, or hybridomas, are cultured, and the resulting colonies screened for the production of the desired monoclonal antibodies. Colonies producing such antibodies are cloned and grown either in vivo or in vitro to produce large quantities of antibody. A description of the theoretical basis and practical methodology of fusing such cells is set forth in Kohler and Milstein, (1975).

Mammalian lymphocytes are immunized by in vivo immunization of the animal (e.g., a mouse) with the protein or polypeptide of the present invention. Such immunizations are repeated as necessary at intervals of up to several weeks to obtain a sufficient titer of antibodies. Following the last antigen boost, the animals are sacrificed and spleen cells removed.

Fusion with mammalian myeloma cells or other fusion partners capable of replicating indefinitely in cell culture is effected by standard and well-known techniques, for example, by using polyethylene glycol (“PEG”) or other fusing agents (See Milstein and Kohler, (1976). This immortal cell line, which is preferably murine, but may also be derived from cells of other mammalian species, including but not limited to rats and humans, is selected to be deficient in enzymes necessary for the utilization of certain nutrients, to be capable of rapid growth, and to have good fusion capability. Many such cell lines are known to those skilled in the art, and others are regularly described.

Procedures for raising polyclonal antibodies are also well known. Typically, such antibodies can be raised by administering the protein or polypeptide of the present invention subcutaneously to New Zealand white rabbits which have first been bled to obtain pre-immune serum. The antigens can be injected at a total volume of 100 ml per site at six different sites. Each injected material will contain adjuvants with or without pulverized acrylamide gel containing the protein or polypeptide after SDS-polyacrylamide gel electrophoresis. The rabbits are then bled two weeks after the first injection and periodically boosted with the same antigen three times every six weeks. A sample of serum is then collected 10 days after each boost. Polyclonal antibodies are then recovered from the serum by affinity chromatography using the corresponding antigen to capture the antibody. This and other procedures for raising polyclonal antibodies are disclosed in Harlow, (1988).

In addition to utilizing whole antibodies, the processes of the present invention encompass use of binding portions of such antibodies. Such binding portions include Fab fragments, F(ab′)2 fragments, and Fv fragments. These antibody fragments can be made by conventional procedures, such as proteolytic fragmentation procedures, as described in Goding, (1983).

The present invention also provides a method of reducing an insect's life span. The insect is contacted with an isolated Drosophila insect toxin protein under conditions effective shorten the insect's life span. In a preferred embodiment, the isolated Drosophila insect toxin protein used to shorten the life span of the insect has an amino acid sequence corresponding to SEQ ID NO:4.

The active ingredients of the present invention are normally applied in the form of compositions and can be applied to the crop area or plant to be treated, simultaneously or in succession, with other compounds. These compounds can be both fertilizers or micronutrient donors or other preparations that influence plant growth. They can also be selective herbicides, insecticides, fungicides, bactericides, nematicides, mollusicides or mixtures of several of these preparations, if desired, together with further agriculturally acceptable carriers, surfactants or application-promoting adjuvants customarily employed in the art of formulation. Suitable carriers and adjuvants can be solid or liquid and correspond to the substances ordinarily employed in formulation technology, e.g. natural or regenerated mineral substances, solvents, dispersants, wetting agents, tackifiers, binders or fertilizers. Preferred methods of applying an active ingredient of the present invention or an agrochemical composition of the present invention which contains at least one of the pesticidal proteins produced by the bacterial strains of the present invention are leaf application, seed coating and soil application. The number of applications and the rate of application depend on the intensity of infestation by the corresponding pest.

In an alternative embodiment of the invention, the gene encoding the insect toxin protein may be introduced into plants. The gene is under the control of a promoter which is effective in plants, so that the insect toxin gene is expressed in the plant. The insects would ingest the protein when eating any portion of the plant. When utilizing transgenic plants, this involves providing a transgenic plant transformed with a DNA molecule encoding a fragment of a insect toxin protein, which fragment is toxic to the target insect, and growing the plant under conditions effective to permit that DNA molecule to control insects. Alternatively, a transgenic plant seed transformed with a DNA molecule encoding a fragment of a hypersensitive response elicitor polypeptide or protein which fragment is toxic to the target insects can be provided and planted in soil. A plant is then propagated from the planted seed under conditions effective to permit that DNA molecule to control insects.

In addition to contacting the insect with the protein of the invention, the invention provides a method of reducing an insect's life span by contacting the insect with an expression vector containing a nucleic acid molecule encoding a Drosophila insect toxin protein under conditions effective to express the protein. In a preferred embodiment, the Drosophila insect toxin protein is a protein having an amino acid sequence according to SEQ ID NO:4, or a protein encoded by a nucleic acid molecule which hybridizes under stringent conditions to a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO:2 and where the protein has the biological function of an insect toxin.

The present invention also provides several other accessory gland proteins and the genes encoding the proteins. As discussed above, the genes are named Acp followed by their location on the polytene chromosomes as determined by in situ hybridization. For example, the site for Acp76A is determined as 75F/76A, for 63F is 63F/64A, for 31F is 31F/32A. The genomic clone containing Acp33A has a minor site of hybridization at 32E. Eight of the proteins predicted from these sequences begin with putative secretion signals. Following their signal sequences, three of the predicted molecules are peptides and the other five are larger polypeptides with characteristics of cleavable prohormones. The ninth molecule, which has an N-terminal hydrophobic region but no consensus signal cleavage site, is predicted to be a 716 amino acid glycoprotein. Of the 9 proteins, two have intriguing similarities to sequences in protein databases. Acp76A is a 388 amino acid pro-protein which contains a signature sequence for the serpin class of protease inhibitors.

The invention provides the isolated nucleic acid molecules which encode a number of novel accessory gland proteins. The preferred embodiments of the invention are nucleic acid molecules having a nucleotide sequence of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:13, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:27, or SEQ ID NO:30. The present invention also includes nucleic acid molecules which hybridizes under stringent conditions to a nucleic acid molecule having a nucleic acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:13, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:27, or SEQ ID NO:30. As above, the invention provides expression vectors and host cells containing the nucleic acids of the invention.

In a preferred embodiment, the isolated nucleic acid molecule encoded a protein having an amino acid sequence corresponding to SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, or SEQ ID NO:32.

Preferred nucleic acid molecules are deoxyribonucleic acid molecules.

The present invention also provides isolated proteins encoded by the accessory gland proteins. Preferred proteins have the amino acid sequences, or fragments thereof, provided in SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, or SEQ ID NO:32.

The present invention provides a gene identified as Acp29AB, its cDNA sequence is as follows (SEQ ID NO:5): 1 gacacgcttg aaatcttcca aactggacat gtacgcatct aacctcttat acctgttggc 61 attatggaac ctttgggatc tcagtggtgg gcagcaggac attccgaacg gaaaggctac 121 attgccaagt ccacaaacgc cgcaaaatac aatcgatcag attggtatta accagaatta 181 ttggtttaca tacaacgcgc ttaaacaaaa cgaaacattg gcaattattg atacaatgga 241 aatgcgcata gcaagtagct tgctggagtt taaggcccag atggaaatcc agcttcagcc 301 gttaaagatt ataatgcgac accatgcatc caacatcaaa gcgtctaaca acatcaagat 361 gagacgattc gagaaagttg gctccagaca ttttcacatc gagaagaatc taatgcaaac 421 ttggtttgag gcatatgtca catgtcgtaa aatgaacggt catctggcga acatccagga 481 tgagaaggag ctggatggca tcttggcgtt agcacccaac aatagctact ggatagatat 541 atccaaactg gttgaaaatg gcggcacatt cgtctccacc ctaaccggac gagaaccctt 601 ctttgttaaa tggaagagta atcaggatac aaaaaaaaag aatcaatgcg tttacatcta 661 tgctaaagag atgtcctatg atgagtgttt tgaaaaaaaa tctttcgttt gccaagcaga 721 ccagtgggcc taaacataaa gaaaatattg ttttgtagct tgaataataa aattataaaa 781 aaaaaaaaaa aa

The cDNA encodes an open reading frame which is located at nucleotides 29 to 733 of SEQ ID NO:5 (SEQ ID NO:6): 1 atgtacgcat ctaacctctt atacctgttg gcattatgga acctttggga tctcagtggt 61 gggcagcagg acattccgaa cggaaaggct acattgccaa gtccacaaac gccgcaaaat 121 acaatcgatc agattggtat taaccagaat tattggttta catacaacgc gcttaaacaa 181 aacgaaacat tggcaattat tgaccagaat taaatgcgca tagcaagtag cttgccggag 241 tttaaggccc agatggaaat ccagcttcag ccgttaaaga ttataatgcg acaccatgca 301 tccaacatca aagcgtctaa caacatcaag atgagacgat tigagaaagt tggctccaga 361 cattttcaca tcgagaagaa tctaatgcaa acttggtttg aggcatatgt cacatgtcgt 421 aaaatgaacg gtcatctggc gaacatccag gatgagaagg agctggatgg catcttggcg 481 ttagcaccca acaatagcta ctggatagat atatccaaac tggttgaaaa tggcggcaca 541 ttcgtctcca ccctaaccgg acgagaaccc ttctttgtca aacggaagag taatcaggat 601 acaaaaaaaa agaatcaatg cgtttacatc tatgctaaag agatgtccta tgatgagtgt 661 tttgaaaaaa aatctttcgt ttgccaagca gaccagtggg cc

The amino acid sequence of the Acp29AB protein is provided as follows (SEQ ID NO:7): 1 MYASNLLYLL ALWNLWDLSG GQQDIPNGKA TLPSPQTPQN TIDQIGINQN YWFTYNALKQ 61 NETLAIIDTM EMRIASSLLE FKAQMEIQLQ PLKIIMRHHA SNIKASNNIK MRRFEKVGSR 121 HFHIEKNLMQ TWFEAYVTCR KMNGHLANIQ DEKELDGILA LAPNNSYWID ISKLVENGGT 181 FVSTLTGREP FFVKWKSNQD TKKKNQCVYI YAKEMSYDEC FEKKSFVCQA DQWA

The invention further provides a gene encoding an accessory gland protein identified as Acp32CD. The cDNA sequence for Acp32CD is as follows (SEQ ID NO:8): 1 cagctgcgct gttgcttcag ctgactgcag caacatggac taacaactgc cactgttttt 61 cagttcccca gatatgcccc ccttgttgcg gcactgcttt ggacacgcct ttatcggact 121 gccccttttt aatgggcaag agcagccacg gccacagtca aatcgattcg attccgggca 181 acggcggtca tctctatata taagggacgg tcggacagcc cgggcagctc agcgttgcag 241 cgatgtggcg gatgcggatg cggctactca ctggctactt ggtcctgtgg ccctcggcca 301 actgccggag catggcgctc tgggccagaa gtactacatg aactttgcct tcaataataa 361 taatccggat ggcgaaggag gcaccggcgt cgatggtggc ggtggtggtg ctggtggtgg 421 tgccgctggt cccggcggtg gaaccgggga ttcgccccat tcacaagaag gtgacggtag 481 tgctgcgacg gataacccca atgacgacca cgctacatct gctgataata gtttggctac 541 tgatggcgat gccattggta agaaggaaag cggcggtggt agcgatggca agagcgactc 601 caaagactcg agcggaggca atgatgccac gccagcgaat ggtcatgacg atgacaacga 661 tgacagcgac agaaggatgc caaggatcga caagataagg aagaggaggc cggacaggag 721 gggaagcgca ccgatcacag ccatcacagt agctacgaga tcagatcgac gacagctttg 781 gcgggcggta cgtgcggtcc atttacgaga gcagcgagag ccacggacat tcggcagcaa 841 tgccggctcc aatcagcgga caatggagcc cgtgagagca gtcaggagaa ccaggatgcc 901 aaggaagtgg ccagcgaagc gcctgctcaa cgggcaggca acgtgccaga tggacccgaa 961 actggagcca accgggagat ctgaccacag gttcgctcga gggactttgg aagaacacag 1021 acacttcaat tgattttatc attaaaatgc caataaaatt ttaataccaa aaaaaaaaaa 1081 aaaaa

Two open reading frames are found within the nucleotide sequence of the Acp32CD cDNA. Open reading frames 1 and 2 are provided as SEQ ID NOS:9 and 10, respectively, as follows: 1 atgcccccct tgttgcggca ctgctttgga cacgccttta tcggactgcc cctttttaat (SEQ ID NO:9) 61 gggcaagagc agccacggcc acagtcaaat cgattcgatt ccgggcaacg gcggtcatct 121 ctatatataa gggacggtcg gacagcccgg gcagctcagc gttgcagcga tgtggcggat 181 gcggatgcgg ctactcactg gctacttggt cctgtggccc tcggccaact gccggagcat 241 ggcgctctgg gccagaagta ctacatgaac tttgccttca acaacaataa tccggatggc 301 gaaggaggca ccggcgtcga tggtggcggt ggtggtgctg gtggtggtgc cgctggtccc 361 ggcggtggaa ccggggattc gccccattca caagaaggtg acggtagtgc tgcgacggat 421 aaccccaatg acgaccacgc tacatctgct gataatagtt tggctactga tggcgatgcc 481 attggtaaga aggaaagcgg cggtggtagc gatggcaaga gcgactccaa agactcgagc 541 ggaggcaatg atgccacgcc agcgaatggt catgacgatg acaacgatga cagcgacaga 601 aggatgccaa ggatcgacaa gataaggaag aggaggccgg acaggagggg aagcgcaccg 661 atcacagcca tcacagtagc tacgagatca gatcgacgac agctttggcg ggcggtacgt 721 gcggtccatt tacgagagca gcgagagcca cggacattcg gcagcaatgc cggctccaat 781 cagcggacaa tggagcccgt gagagcagtc aggagaacca ggatgccaag gaagtggcca 841 gcgaagcgcc tgctcaacgg gcaggcaacg tgccagatgg acccgaaact ggagccaagg 901 aagatgacta cgaggagatg taacacaacc gggagatctg accacaggtt cgctcgaggg 961 actttggaag aacgcagaca cttcaat

1 atgaactttg ccttcaataa taataatccg gatggcgaag gaggcaccgg cgtcgatggt (SEQ ID NO:10) 61 ggcggtggtg gtgctggtgg tggtgccgct ggtcccggcg gtagaaccgg ggattcgccc 121 cattcacaag aaggtgacgg tagtgctgcg acggataacc ccaatgacga ccacgctaca 181 tctgctgata atagtttggc tactgatggc gatgccattg gtaagaagga aagcggcggt 241 ggtagcgatg gcaagagcga ctccaaagac tcgagcggag gcaatgatgc cacgccagcg 301 aatggtcatg acgatgacaa cgatgacagc gacagaagga tgccaaggat cgacaagata 361 aggaagagga ggccggacag gaggggaagc gcaccgatta cagccatcac agtagctacg 421 agatcagatc gacgacagct ttgtgccggc tccaatcagc ggacaatgga gcccgtgaga 481 gcagtcagga gaaccaggat gccaaggaag tggccagcga agcgcctgct caacgggcag 541 gcaacgtgcc agatggaccc gaaactggag ccaaggaaga tgactacgag gagatgtaac 601 acaaccggga gatctgacca caggttcgct cgagggactt tggaagaacg cagacacttc 661 aat

Open reading frame 1, encodes a protein of 269 amino acids. The predicted amino acid sequence from open reading frame 1 of the ACP32CD gene is provided in SEQ ID NO:11, as follows: 1 MPPLLRHEFG HAFIGLPLFN GQEQPRPQMN FAFNNNNPDG EGGTGVDGGG GGAGGGAAGP 61 GGGTGDSPHS QEGDGSAATD NPNDDHATSA DNSLATDGDA IGKKESGGGS DGKSDSKDSS 121 GGNDATPANG HDDDNDDSDR RMPRIDKIRK RRPDRRGSAP ITAITVATRS DRROLWRAVR 181 AVHLREOREP RTFGSNAGSN QRTMEPVRAV RRTRMPRKWP AKRLLNGQAT CQMDPKLEPR 241 KMTTRRCNTT GRSDHRFARG TLEERRHFN

Open reading frame 2, encodes a shorter protein lacking the amino terminal 28 amino acids of the protein encoded by open reading frame 1. The predicted amino acid sequence from open reading frame 2 of the ACP32CD gene is provided in SEQ ID NO:12, as follows: 1 MNFAFNNNNP DGEGGTGVDG GGGGAGGGAA GPGGGTGDSP HSQEGDGSAA TDNPNDDHAT 61 SADNSLATDG DAIGKKESGG GSDGKSDSKD SSGGNDATPA NGHDDDNDDS DRRMPRIDKI 121 RKRRPDRRGS APITAITVAT RSDRROLWRA VRAVHLREOR EPRTEGSNAG SNQRTMEPVR 181 AVRRTRMPRK WPAKRLLNGQ ATCQMDPKLE PRKMTTRRCN TTGRSDHRFA RGTLEERRHF

The present invention also provides a gene encoding an accessory gland protein which is identified as Acp33A (SEQ ID NO:13): 1 ggattctaca agatgctacc ttccaagcga gttccatttc ttttcaccat tatcctgttt 61 ctggctggac tgggtcagca cacaactgaa agtgtacttc cagactgcgt tctttatcca 121 agatgtttga tcacaaagga tccgtgttgc atgtaaagca tctatagaaa tcaaagaagg 181 atcttgtata tggaagtgga aatgtggttc aaaatatcag cacttctcaa ggctgtaaca 241 aaaactgcac tctgttttta taaatacaaa tatccacaaa tatctggtct taacatggca 301 acatttcaag tcttccaata aatcattttc gtttttattg tgaaaaaaaa aaaaaaaaaa 361 aaaaaaa

Two open reading frames are also found within the nucleotide sequence of the Acp32CD cDNA. Open reading frames 1 and 2 are provided as SEQ ID NOS:14 and 15, respectively, as follows: 1 atgctacctt ccaagcgagt tccatttctt ttcaccatta tcctgtttct ggctggactg (SEQ ID NO:14) 61 ggtcagcaca caactgaaag tgtacttcca gactgcgttc gactgcgttc tttatccaag 121 atgtttga tcacaaagga tccgtgttgc atg

1 atggaagtgg aaatgtggtt caaaatatca gcacttctca aggctgtaac aaaaactgca (SEQ ID NO:15) 61 ctctgttttt ataaatacaa atatccacaa atatctggtc ttaacatggc aacatttcaa 121 gtcttccaa

Open reading frame 1, encodes a protein of 47 amino acids. The predicted amino acid sequence from open reading frame 1 of the ACP32CD gene is provided in SEQ ID NO:16, as follows: 1 MLPSKRVPFL FTIILFLAGL GQHTTESVLP DCVLYPRCLI TKDPCCM

Open reading frame 2, encodes a protein of 43 amino acids. The predicted amino acid sequence from open reading frame 2 of the ACP32CD gene is provided in SEQ ID NO:17, as follows: 1 MEVEMWFKIS ALLKAVTKTA LCFYKYKYPQ ISGLNMATFQ VFQ

The present invention also provides a gene encoding an accessory gland protein which is Acp36DE (SEQ ID NO:18): 1 caaatcaaca actaattaat gcacaagacg tgctctcaga caaagatcag aaacagacgc 61 aagttcagaa caataactta catattcgat tcggtgtgtc agcactaaga gaaggaagaa 121 ataatcctag tttggaaacg atttctcggg ataaagtaga taaaatatca cctgcattgc 181 agttgcaact gttgagatat gcagattctc agtcgcaatc ccagacgcag tcacaatctg 241 cctcacaatc tgaatcaaat gcatcttcac aattccaggc acaggagcaa agcaatcgac 301 tgttgggaaa acccacctgt ttcagaatct cagtcacaat cagagtcaca gtcacagtcc 361 gagtcacaga agcagtcaca gtcgcagtca cagcgacagc aacagataca gacgcaattg 421 caaatactgc gacagttgca acaaaagtca aatgagcaat ctgccgcaca atctgcttct 481 cagattcaat cgcagaggca atcggattct caatccaact tacaattaca agaacaatca 541 caatcgcagt cagagcaagg taagccaatc cagtcacaaa ttcaaattct tcaagggctg 601 cagcaaaaag agttagatga caaatctgca tcacagtcgc agtccgaatc caagacacgg 661 aaagagcaac aaaaacagtt gaatttgcaa caacttgagg agctatcgtc ttcactatca 721 cagtcacggc tagggctggg acagcaaatc cagtcacagc tacaaaagaa tcagttggat 781 aagcaatttt cttcacagtt tcagtcacaa tccaagtcac agctggagca acaaatgcaa 841 ttgcaattac aaagccttcg gcaactgcag cagaagcaat tagatgagca atctgcttca 901 cagtcgcagc cacagtcaca ggtagcgcaa cagatccagt cacatttgca acttcttcga 961 ttactgcaat ccagattgaa gacgcagtcg gcattgaaat cagatttaga acaacaaatc 1021 ctttttcaat taaagaaact tacagaagtg caacagaaac agttggctga gcaacccacc 1081 ttacgaccca gttcaaaatc acaatcgcct gggcagctag agcagcaaat tctgttacac 1141 ctgcaaaatc ttctacactt tcagcagaat cagctaaaat cagatacaca aacccaaagc 1201 cagttgcaag agtcaaaatc taactcactg tcacagtcac agtcacaatc gcaggagcag 1261 ttacagttgc agcgggatca gaatcttcgg caattggaac aagtaaagtt ggaaatgcaa 1321 aatattcgag agctgctgca gaagggcaag tctgagctac aaacccaatc ggactctcag 1381 cgacgtatac atgagctata ccaaaatatt ctgcagctaa ataaggagaa gttgagctac 1441 caattgaaac agttaaaact aaaagaattg gaagaccaaa agaagtcgca ggcagaaata 1501 tcaaagggaa gtaacccatc caatctattt attatcggac aattgccttc cgaaggaaag 1561 ccagctcctg gaaatcaagg tccttcaatt gagcctaagc tggtccccca acccggttca 1621 ctggacaaat tgccatcagg cggagggcta attggcaagc cagcttcaac aggactgtat 1681 attttatcgc cggatttcaa tgatttgtcg gattaccgag atcagtttcg tctacagcaa 1741 gaattaaaaa agcatcaaaa tatattgagc cttttgcagc gtagacaaaa tgataaaaaa 1801 caacaaaacg cacagctgtt gctaggacaa caacagaagg aacaacaagc tcaggaatca 1861 atcaataaac aacagtcctc atctgctggc tctagttctc agaccaagtt acagcaagat 1921 atacaaagta ctggagctca aggctcacag cagggtcttc aagctggatc cactggcctg 1981 cagactagtt ccctacaagg cacagaaagt tctgcatctc aaagcgctct tcagcgattg 2041 aaggagcagg aacaactgcg aattcagacg gaaaatgatc agaaaacctc ttcttcaagc 2101 tcgcacagta actcacaaaa ctcgcagagt tcgtcatcac agtcatcgca ggcatcacag 2161 tctgaagcac aacgacagga ggctggcaat cgaaatacct tgctactaga tcaatcgagc 2221 tccaagactc agtcgagtcg aagtccgagt cgtcgtctca atcatcgtca cattcatcgt 2281 cgcagtcaac gtcgaactca tcttcaaacg ttcaatcgaa actacaagga gaaagccaag 2341 cgctgctaaa caatttgtca ggttaagtag ttaaccttat acttctcaca gtactgacat 2401 gggcgaagag cagccttatt cagatgttaa tcaaaaagag gaaataaaat aattgttctt 2461 tcatttaaaa actcgaaaaa aaaaaaaaaa aa

An open reading frame encoding the Acp36DE protein is found within the cDNA sequence (SEQ ID NO:19): 1 atgcagattc tcagtcgcaa tcccagacgc agtcacaatc tgcctcacaa tctgaatcaa 61 atgcatcttc acaattccag gcacaggagc aaagcaatcg actgttggga aaacccacct 121 gtttcagaat ctcagtcaca atcagagtca cagtcacagt ccgagtcaca gaagcagtca 181 cagtcgcagt cacagcgaca gcaacagata cagacgcaat tgcaaatact gcgacagttg 241 caacaaaagt caaatgagca atctgccgca caatctgctt ctcagattca atcgcagagg 301 caatcggatt ctcaatccaa cttacaatta caagaacaat cacaatcgca gtcagagcaa 361 ggtaagccaa tccagtcaca aattcaaatt cttcaagggc tgcagcaaaa agagttagat 421 gacaaatctg catcacagtc gcagtccgaa tccaagacac ggaaagagca acaaaaacag 481 ttgaatttgc aacaacttga ggagctatcg tcttcactat cacagtcacg gctagggctg 541 ggacagcaaa tccagtcaca gctacaaaag aatcagttgg ataagcaatt ttcttcacag 601 tttcagtcac aatccaagtc acagctggag caacaaatgc aattgcaatt acaaagcctt 661 cggcaactgc agcagaagca attagatgag caatctgctt cacagtcgca gccacagtca 721 caggtagcgc aacagatcca gtcacatttg caacttcttc gattactgca atccagattg 781 aagacgcagt cggcattgaa atcagattta gaacaacaaa tcctttttca attaaagaaa 841 cttacagaag tgcaacagaa acagttggct gagcaaccca ccttacgacc cagttcaaaa 901 tcacaatcgc ctgggcagct agagcagcaa attctgttac acctgcaaaa tcttctacac 961 tttcagcaga atcagctaaa atcagataca caaacccaaa gccagttgca agagtcaaaa 1021 tctaactcac tgtcacagtc acagtcacaa tcgcaggagc agttacagtt gcagcgggat 1081 cagaatcttc ggcaattgga acaagtaaag ttggaaatgc aaaatattcg agagctgctg 1141 cagaagggca agtctgagct acaaacccaa tcggactctc agcgacgtat acatgagcta 1201 taccaaaata ttctgcagct aaataaggag aagttgagct accaattgaa acagttaaaa 1261 ctaaaagaat tggaagacca aaagaagtcg caggcagaaa tatcaaaggg aagtaaccca 1321 tccaatctat ttattatcgg acaattgcct tccgaaggaa agccagctcc tggaaatcaa 1381 ggtccttcaa ttgagcctaa gctggtcccc caacccggtt cactggacaa attgccatca 1441 ggcggagggc taattggcaa gccagcttca acaggactgt atattttatc gccggatttc 1501 aatgatttgt cggattaccg agatcagttt cgtctacagc aagaattaaa aaagcatcaa 1561 aatatattga gccttttgca gcgtagacaa aatgataaaa aacaacaaaa cgcacagctg 1621 ttgctaggac aacaacagaa ggaacaacaa gctcaggaat caatcaataa acaacagtcc 1681 tcatctgctg gctctagttc tcagaccaag ttacagcaag atatacaaag tactggagct 1741 caaggctcac agcagggtct tcaagctgga tccactggcc tgcagactag ttccctacaa 1801 ggcacagaaa gttctgcatc tcaaagcgct cttcagcgat tgaaggagca ggaacaactg 1861 cgaattcaga cggaaaatga tcagaaaacc tcttcttcaa gctcgcacag taactcacaa 1921 aactcgcaga gttcgtcatc acagtcatcg caggcatcac agtctgaagc acaacgacag 1981 gaggctggca atcgaaatac cttgctacta gatcaatcga gctccaagac tcagtcgagt 2041 cgaagtccga gtcgtcgtct caatcatcgt cacattcatc gtcgcagtca acgtcgaact 2101 catcttcaaa cgttcaatcg aaactacaag gagaaagcca agcgctgc

The predicted amino acid sequence of the Acp36DE protein is as follows (SEQ ID NO:20): 1 MQILSRNPRR SHNLPHNLNQ MHLHNSRHRS KAIDCWENPP VSESQSQSES QSQSESQKQS 61 QSQSQRQQQI QTQLQILRQL QQKSNEQSAA QSASQIQSQR QSDSQSNLQL QEQSQSQSEQ 121 GKPIQSQIQI LQGLQQKELD DKSASQSQSE SKTRKEQQKQ LNLQQLEELS SSLSQSRLGL 181 GQQIQSQLQK NQLDKQFSSQ FQSQSKSQLE QQMQLQLQSL RQLQQKQLDE QSASQSQPQS 241 QVAQQIQSHL QLLRLLQSRL KTQSALKSDL EQQILFQLKK LTEVQQKQLA EQPTLRPSSK 301 SQSPGQLEQQ ILLHLQNLLH FQQNQLKSDT QTQSQLQESK SNSLSQSQSQ SQEQLQLQRD 361 QNLRQLEQVK LEMQNIRELL QKGKSELQTQ SDSQRRIHEL YQNILQLNKE KLSYQLKQLK 421 LKELEDQKKS QAEISKGSNP SNLFIIGQLP SEGKPAPGNQ GPSIEPKLVP QPGSLDKLPS 481 GGGLIGKPAS TGLYILSPDF NDLSDYRDQF RLQQELKKHQ NILSLLQRRQ NDKKQQNAQL 541 LLGQQQKEQQ AQESINKQQS SSAGSSSQTK LQQDIQSTGA QGSQQGLQAG STGLQTSSLQ 601 GTESSASQSA LQRLKEQEQL RIQTENDQKT SSSSSHSNSQ NSQSSSSQSS QASQSEAQRQ 661 EAGNRNTLLL DQSSSKTQSS RSPSRRLNHR HIHRRSQRRT HLQTFNRNYK EKAKRC

The present invention provides a gene encoding an accessory gland protein which is Acp53Ea (SEQ ID NO:21): 1 ccgaaagcac acagataagg cttccaatga aactgataaa ggttacacta gtgttcagct 61 tactggctct cgtatttgtg gcccaaacgg aggcgcaaaa tccaatatgg gagaattggc 121 tggcatgcaa tagaattggt actaaagcgc ttgccagtct gctgagagaa acaattccaa 181 ccgttcgtaa tttactgaac tgcattgact tcaatccacc aaccgatatt ggaaatagtt 241 acctttcaaa acttaagtta tactatgagc ttgttaagcg aggtgcgctt gacaagactc 301 agtgtctgat tgtgccactc aaggaatcag tgagactact gaggccttat gtaaaatcgc 361 ttgagaccaa caaatgcttg ggtgaataaa tcactatttt ggccatagta aaataaattt 421 ctgagcatta ataaagcacg

An open reading frame encoding the Acp53Ea protein is found within the cDNA sequence (SEQ ID NO:22): 1 atgaaactga taaaggttac actagtgttc agcttactgg ctctcgtatt tgtggcccaa 61 acggaggcgc aaaatccaat atgggagaat tggctggcat gcaatagaat tggtactaaa 121 gcgcttgcca gtctgctgag agaaacaatt ccaaccgttc gtaatttact gaactgcatt 181 gacttcaatc caccaaccga tattggaaat agttaccttt caaaacttaa gttatactat 241 gagcttgtta agcgaggtgc gcttgacaag actcagtgtc tgattgtgcc actcaaggaa 301 tcagtgagac tactgaggcc ttatgtaaaa tcgcttgaga ccaacaaatg cttgggtgaa

The predicted amino acid sequence of the Acp53Ea protein is as follows (SEQ ID NO:23): 1 MKLIKVTLVF SLLALVFVAQ TEAQNPIWEN WLACNRIGTK ALASLLRETI PTVRNLLNCI 61 DFNPPTDIGN SYLSKLKLYY ELVKRGALDK TQCLIVPLKE SVRLLRPYVK SLETNKCLGE

The present invention provides a gene encoding an accessory gland protein which is identified as Acp63F (SEQ ID NO:24): 1 ctttgcaaga tgaaagctat catcgttttt attctgttca tttcaagtgt gcatgctatg 61 agcaaatgca accaagcaat ttatctaaat cttgatcctc actgcggaat acttcccgat 121 tgtaacttag atggtccaaa tccaagttac ctcaataggg tgtcgtgtga acgcaaagaa 181 aacggaaaac caggattcat cgaactaatt cccggaaaat gtctccatgg taaaccgcgt 241 tgctcgttaa aatagtaata ttgttccaat atttccatgc atatatgttt caattaaagg 301 cattataaat acctataaaa aaa

An open reading frame encoding the Acp63F protein is found within the cDNA sequence (SEQ ID NO:25): 1 atgaaagcta tcatcgtttt tattctgttc atttcaagtg tgcatgctat gagcaaatgc 61 aaccaagcaa tttatctaaa tcttgatcct cactgcggaa tacttcccga ttgtaactta 121 gatggtccaa atccaagtta cctcaatagg gtgtcgtgtg aacgcaaaga aaacggaaaa 181 ccaggattca tcgaactaat tcccggaaaa tgtctccatg gtaaaccgcg ttgctcgtta 241 aaa

The predicted amino acid sequence of the Acp63F protein is as follows (SEQ ID NO:26): 1 MKAIIVFILF ISSVHAMSKC NQAIYLNLDP HCGILPDCNL DGPNPSYLNR VSCERKENGK 61 PGFIELIPGK CLHGKPRCSL K

The present invention provides a gene encoding an accessory gland protein which is identified as Acp76A (SEQ ID NO:27): 1 atatcaagct tatgactctt ggataagcca ttgatatagc aattgtaaat atattatgtg 61 caattctcct tattttctaa gacattctta ataatataat gcgaactaag gttacattcc 121 atctgcgcat gcgtgagtcc ttttgctcag caggaacgaa agtacaacgg gtcgtatgag 181 ttatggcaat cgaatgggca cacgtggccg ccggtccaat ggccagtaaa tcaaactttt 241 tggcagggcg agtaaacagt gttataaatc agaaaaccgc aaggcagcca ccaggcagct 301 accatattgt ccatggagga aagcagacag ttggcagact taagtcggac gaaaagacat 361 ccacccacgc gggcggattg caatgtgcca ggatgcaagt gacgcatagt gctattaaca 421 tattaccagg cggtaagtgg gtggataaca cattcagtcg gtggatggaa gtacgtgcat 481 agaacataga gctgccagtg aatattggag caattggagc actgggtgct aaatatgagc 541 acttgatgaa aacataacac tgactaaaca agatatttct ctgcggctga atttatctag 601 cgaaagtgtg aaattgttgt tgatttatgt tatactaagc acgccatata tatgaggtgg 661 cgaatttacg acgatttact aagattttaa tgtactcttc cttggttaga ggatagaaag 721 catgaaattg aatagcagtg caataaatcc gtactaattt ccattcgttt ttgcgtatta 781 tccaatatct taagggtcat tccccttgtg tgtatatata ggaacaaagt aagtttaaga 841 agctcattgc ggctttggaa aatgggcaac catcaagtaa cattcttagt actgtgcacg 901 tcgctcctct ttcaaaatac aatacaacaa aatgtatcat ttcaactgat aagggaaatc 961 gatagataca caccagagaa ttttgtacta tcagtgttga atatagaaat gattcttttt 1021 gagatccatg ccgctaaggc agttgaaagt aataacgatt tggaaaggag cttgatcata 1081 aactttggat actccgaagc aaggcaggaa gtactggatt ggggattgag atataagaaa 1141 gcctcgagcg ccaagttcca gatggccaac aaggtggcag tgtctcagaa actgccccta 1201 tcgcaaaagc tgcgtctggt aaacgaggtg ctgatgacga gcgccaagaa gtatgatgta 1261 acaaaggatg tcagaccatc aaaattaatg gatgaatggt tgtcctccca tttggatggt 1321 gtactcgcca attttgtaca agagaagaag ttaaacgcgg gcgaaaacat tgtagccatc 1381 agcggaatga cagtcactcc cctttgggca tctcatttcc aatcagagat taatagatac 1441 tttgtcaata atcctggcac tggatatgct tcgaaagacc caacatgtgt gcccatgatg 1501 cactcattgt cctcgtttga aaccatgtcc acggacgagg ccaaaggtat atacattcca 1561 ttctcatcgg caaacttggg tatgttgatc ctcctgccga ggaaaggtgt cacctgcaag 1621 gacattttgg ataatttaaa caaccagatc aatgtggaat ataatgatca caaggatgtt 1681 cacttgctac tgcccatatt caaggagaaa tttgactaca atattgccaa attctttaac 1741 ggaattaaca ttgaagacac gtttaaagat tcggcgttta aatcgaaagc caaaatcaaa 1801 atcaacaact tccgagtcaa ccatggcata cgatttcaac ccattctccg tttagaagta 1861 gttgatgata ttaatactgg aaagaccgaa acgtttgaag taaatcgccc atttgtcttt 1921 gtcataaagg ataaggttaa cgtatacgca gttggtcgaa ttgaaaacct agatggactt 1981 actgacaaag tgaattgctc caagaaatac gctgatctca agtcgtaaaa tatccataat 2041 atatttcgaa gcataataaa gcaagaaaat ataaaaaaaa aaaaaaaaaa aa

An open reading frame encoding the Acp76A protein is found within the cDNA sequence (SEQ ID NO:28): 1 atgggcaacc atcaagtaac attcttagta ctgtgcacgt cgctcctctt tcaaaataca 61 atacaacaaa atgtatcatt tcaactgata agggaaatcg atagatacac accagagaat 121 tttgtactat cagtgttgaa tatagaaatg attctttttg agatccatgc cgctaaggca 181 gttgaaagta ataacgattt ggaaaggagc ttgatcataa actttggata ctccgaagca 241 aggcaggaag tactggattg gggattgaga tataagaaag cctcgagcgc caagttccag 301 atggccaaca aggtggcagt gtctcagaaa ctgcccctat cgcaaaagct gcgtctggta 361 aacgaggtgc tgatgacgag cgccaagaag tatgatgtaa caaaggatgt cagaccatca 421 aaattaatgg atgaatggtt gtcctcccat ttggatggtg tactcgccaa ttttgtacaa 481 gagaagaagt taaacgcggg cgaaaacatt gtagccatca gcggaatgac agtcactccc 541 ctttgggcat ctcatttcca atcagagatt aatagatact ttgtcaataa tcctggcact 601 ggatatgctt cgaaagaccc aacatgtgtg cccatgatgc actcattgtc ctcgtttgaa 661 accatgtcca cggacgaggc caaaggtata tacattccat tctcatcggc aaacttgggt 721 atgttgatcc tcctgccgag gaaaggtgtc acctgcaagg acattttgga taatttaaac 781 aaccagatca atgtggaata taatgatcac aaggatgttc acttgctact gcccatattc 841 aaggagaaat ttgactacaa tattgccaaa ttctttaacg gaattaacat tgaagacacg 901 tttaaagatt cggcgtttaa atcgaaagcc aaaatcaaaa tcaacaactt ccgagtcaac 961 catggcatac gatttcaacc cattctccgt ttagaagtag ttgatgatat taatactgga 1021 aagaccgaaa cgtttgaagt aaatcgccca tttgtctttg tcataaagga taaggttaac 1081 gtatacgcag ttggtcgaat tgaaaaccta gatggactta ctgacaaagt gaattgctcc 1141 aagaaatacg ctgatctcaa gtcg

The predicted amino acid sequence of the Acp76A protein is as follows (SEQ ID NO:29): 1 MGNHQVTFLV LCTSLLFQNT IQQNVSFQLI REIDRYTPEN FVLSVLNIEM ILFEIHAAKA 61 VESNNDLERS LIINFGYSEA RQEVLDWGLR YKKASSAKFQ MANKVAVSQK LPLSQKLRLV 121 NEVLMTSAKK YDVTKDVRPS KLMDEWLSSH LDGVLANFVQ EKKLNAGENI VAISGMTVTP 181 LWASHFQSEI NRYFVNNPGT GYASKDPTCV PMMHSLSSEE TMSTDEAKGI YIPFSSANLG 241 MLILLPRKGV TCKDILDNLN NQINVEYNDH KDVHLLLPIF KEKFDYNIAK FFNGINIEDT 301 FKDSAFKSKA KIKINNFRVN HGIRFQPILR LEVVDDINTG KTETFEVNRP FVFVIKDKVN 361 VYAVGRIENL DGLTDKVNCS KKYADLKS

The present invention provides a gene encoding an accessory gland protein which is identified as Acp98AB (SEQ ID NO:30): 1 atggaattcc ctaatcctgt tcttagaaga gcagcaggac attccgaacg gaaaggctac 61 acacactatc aaaggatgac gaggatgtcc aagtgaatca ccagctaaag caaggatatt 121 atgtatatct aaggaaatca aataaacttg catgctctaa aaa

An open reading frame encoding the Acp98AB protein is found within the cDNA sequence (SEQ ID NO:31): 1 atggaattcc ctaatcctgt tcttagccgc attgggcgca gcctccgcac gaataagggg 61 acacactatc aaaggatgac gaggatgtcc aag

The predicted amino acid sequence of the Acp98AB protein is as follows (SEQ ID NO:32): 1 MEFPNPVLSR IGRSLRTNKG THYQRMTRMS K

The present invention also provides a method for determining whether a female Drosophila melanogaster has recently mated. An antibody, fragment thereof, or probe which recognizes an acessory protein, including those proteins described in SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, and SEQ ID NO:32, is used to identify the presence of the accessory gland protein in the female after mating. The antibody, fragment thereof, or probe is bound to a label so that it is effective to permit detection of the protein upon binding of the antibody to the protein. The labeled antibody is contacted with a fluid or tissue sample from the female Drosophila melanogaster under conditions effective to permit binding of the antibody to the protein. The presence of any of protein in the biological sample by is determined by detecting the label.

EXAMPLES Example 1 Materials and Methods

Fly Handling and Rearing

Wild type Canton S or Oregon R D. melanogaster were maintained on yeast-glucose media at room temperature. Unmated animals were anesthetized with CO₂ and collected within 10 hours of eclosion. Flies were kept in fresh vials isolated from the opposite sex until dissected or quick-frozen prior to RNA extraction.

Isolation of New Accessory Gland Genes.

By minor modifications of procedures described in DiBenedetto (1987), 32P-labeled cDNAs were prepared from the poly A+ RNA of 2-day-old adult female Canton S flies and, separately, from accessory glands (attached to ejaculatory ducts) that had been dissected from 2-day-old adult male Canton S flies. These cDNAs were hybridized to duplicate sets of filters containing a total of 22,000 clones of genomic DNA in Charon 4A (Maniatis, 1978). One set of filters received 1.2×10⁷ cpm of labeled cDNA from male reproductive tissues. Prior to incubation with the filters this cDNA was prehybridized with 20 ug of polyA+ RNA from adult females; the female RNA competitor was left in during the filter hybridization. The second set of filters received 1.2×107 cpm of female-derived cDNA, without competitor.

39 clones that consistently showed strong (or exclusive) hybridization to the male tissue probe but not to the female tissue probe through three sequential rounds of plaque purification and screening were retained for further study. These clones were then screened for hybridization to RNAs from accessory glands, or ejaculatory ducts, or testes, or the remainder of the male, or whole adult females. A clone is defined as encoding an accessory gland-specific transcript if that RNA is present in accessory glands but not in any of the other RNA sources listed above. Twenty clones fell into this category. By screening these 20 clones for hybridization to previously cloned accessory gland genes, six re-isolates of the genomic region encoding Acp95EF were found (DiBenedetto, 1987; DiBenedetto, 1990). The remaining 14 clones corresponded to previously-unreported Acp genes. Of these clones, three encompassed overlapping segments of genomic DNA encoding Acp33A; the remaining 11 were single isolates.

Nucleic Acids

The fragment(s) of each genomic clone homologous to the male RNA were identified by Northern and “reverse Northern” strategies. Those fragments were used as probes to isolate cDNA clones from male adult Drosophila melanogaster libraries (Poole, 1985; Monsma and Wolfner, 1988). Routine nucleic acid manipulations including cloning and subcloning into Bluescript vectors (Stratagene), restriction mapping, Southern blotting, determination of transcriptional orientation, DNA sequencing by the dideoxy chain termination method (Sanger, 1977), and isolation of overlapping genomic clones for Acp36DE were done essentially as in Maniatis (1978) and Ausubel (1994). RNA preparation, Northern blotting and hybridizations in situ to polytene chromosomes were by slight modifications of methods described in DiBenedetto (1987). When needed, sequences from multiple clones of a given Acp were aligned to obtain the complete sequence of the open reading frame. In four instances (Acps 32CD, 33A, 76A and 98AB) the cDNA clone was incomplete and lacked the start of the open reading frame and/or any untranslated leader region. Since our focus is on the protein encoded by the Acp gene, RACE-PCR and/or sequencing of genomic DNA immediately 5′ to the first base of the cDNA clone was used to obtain the full open reading frame. This analysis did not extend to defining the starting nucleotide of the Acp mRNA; thus lengths of 5′ UTRs given in FIG. 1 are minimal sizes. Sequences were analyzed with GCG sequence analysis software, and have been submitted to GenBank. Accession numbers are given in FIG. 1.

Whole-mount in Situ Hybridization

Accessory glands were dissected in PBS and treated essentially as in Lehmann and Tautz (1994), except that PBS was used rather than PBST, pretreated the tissue in 50 ug/ml of proteinase K followed by incubation in 2 mg/ml glycine in PBS-Triton, and eliminated heparin from the hybridization buffer. Hybridization and detection with a digoxigenin-labeled probe (Genius Kit, Boehringer Mannheim) was by minor modification of the procedures in Tautz and Pfeifle (1989). Stained accessory glands were mounted on slides in 25% glycerol and photographed using a Zeiss Axioskop and Kodak Gold 200 film.

Example 2 New Genes Encoding Accessory Gland Proteins (Acps)

In differential cDNA hybridization screens, 12 independent new genes were identified (FIG. 1). The genomic clones were restriction-mapped, and their fragment(s) that hybridized to the accessory gland-specific transcript were determined. On Southern blots of genomic DNA, 11 of the genes showed hybridization patterns consistent with single copy genes. All but one of these genes hybridized in situ to a single polytene band; the exception was Acp33A, which showed a minor second site of hybridization at 32E. Each gene's chromosomal position is included in the gene's name in accordance with Drosophila melanogaster nomenclature rules. Two genes, Acp53Ea and Acp53Eb, are derived from polytene band 53E. However they are distinct, non cross-hybridizing genes whose immediate chromosomal regions do not overlap. The genomic clones containing Acp genes did not hybridize to any other male-limited transcripts. However, some of the genomic clones contained regions that hybridized additionally to sex-nonspecific or female-specific transcripts.

The twelfth gene detected in the screens, Acp rep1, had characteristics of a moderately-repetitive gene. Acp rep1 probes hybridized to multiple bands on genomic Southern blots and multiple polytene loci upon in situ hybridization. This gene was not studied further.

On the basis of their chromosomal positions and/or hybridization, these new genes do not correspond to any of the previously-reported D. melanogaster Acp genes identified in hybridization screens or from purified accessory gland peptides (Schäfer, 1986; DiBenedetto, 1987; Chen, 1988; Monsma and Wolfner, 1988; Simmerl, 1995). One of the 11 unique genes, Acp36DE, may correspond to a previously-identified locus, AcpC, which was detected via a natural variant lacking a protein band of ˜125-128 kDa on SDS polyacrylamide gels of accessory gland proteins (Whalen and Wilson, 1986). As described below, Acp36DE encodes a protein of a predicted molecular weight of 81.3 kDa. However, the protein's sequence contains a large number of potential and actual (Bertram, 1996) glycosylation sites, and antibodies against Acp36DE recognize a 122 kDa glycoprotein (Bertram, 1996). Unfortunately, the AcpC variant strains reported by Whalen and Wilson (1986) were no longer extant when Acp36DE was discovered, so it is impossible to determine whether AcpC corresponds to Acp36DE. The approximate genetic map position of one other Acp gene reported by Whalen and Wilson (1986), AcpB, is near Acps 29AB, 31F and 32CD. The other two positions reported by Whalen and Wilson (1986) do not correspond to sites of any of the Acp genes identified.

Thus, 19 non-repetitive Acp genes have been cloned (Schäfer, 1986; DiBenedetto, 1987; Chen, 1988; Monsma and Wolfner, 1988; Simmerl, 1995; and this study). These genes represent 16 independent chromosomal regions; 26A and 57D contain clusters of two (Acp26Aa,b; Monsma and Wolfner, 1988) and three (57Da,b,c; Simmerl, 1995) unrelated Acp genes, respectively.

Interestingly, the new nonrepetitive Acp genes are all autosomal. So are all four Acp genes or gene clusters isolated in previous screens that were unbiased as to chromosomal position (Schäfer, 1986; DiBenedetto, 1987; Chen, 1988). Given that the X chromosome represents about 20% of the genome, the probability that all 15 Acp gene regions would fall outside the X is (0.8)15, which equals 0.035. Even making worst-case assumptions about the library screened (Maniatis, 1978), like its containing no overlapping clones and its deriving from DNA of a 50-50 mixture of XX and XY embryos, it is calculated that p=0.076, which is highly suggestive of nonrandom placement of Acp genes. The absence of Acp genes from the X chromosome might relate to their male-limited expression. In D. melanogaster males, autosomal genes are present in two copies per cell whereas X-linked genes occur in only one copy per cell. Dosage compensation increases the transcription of non sex-specific X-linked genes in male cells so that their transcript levels are equivalent to those produced by two copies of the gene female cells (reviewed in Kelley and Kuroda, 1995). It may be that autosomal placement of Acp genes was advantageous since the genes could be expressed at high levels without also needing to acquire dosage compensation regulation.

Acp genes were first isolated in screens for male-specific transcripts (Schäfer, 1986; DiBenedetto, 1987). Not surprisingly, the screen reported here, with its enriched probe used in the presence of RNA competitor, was 10-fold more efficient than the earlier screens in detecting Acp genes. Considering that from this screen 10 genes were isolated singly, three independent isolates of one were obtained, and six were presumed independent isolates of another, a rough estimate of the number of Acp genes that can be isolated by this type of screen can be made. Since one cannot assume equal representation of all sequences in the library, Bunge and Fitzpatrick (1993) recommend using the method of Esty (1986) for this estimate, even though this estimator is associated with large standard deviations. This estimator [cˆ=c/(1−(c1/n)); Esty, 1986] predicts the existence of 25.3 Acp genes detectable by our screening procedures. The method of Good (1950) predicts about 19 Acp genes detectable by the present procedures. This method assumes all genes are equally represented in the library, based on estimates of “coverage” as defined by Bunge and Fitzpatrick (1993). The Good method tends to yield an underestimate if the assumption of complete coverage is not met. Though these are rough estimates, both calculations suggest that the Acp genes isolated in differential screens performed here and by others represent more than half the genes that could be isolated in such screens, which will fail to detect rare RNAs or Acp genes flanked by highly-expressed non sex-specific transcription units.

Example 3 Expression of the New Acps

Northern blot analysis was performed with each of the Acp genes; an example of the results is shown in FIG. 2A. All the genes with the exception of Acp31F encode single accessory gland-specific transcripts. The transcripts fell into three size classes. Two genes encoded transcripts larger than 2 kb, four encoded mRNAs between 0.95 and 2 kb, and six encoded RNAs smaller than 0.75 kb. The sizes of the transcripts are listed in FIG. 1.

To determine the type of accessory gland cells in which these transcripts are produced, digoxigenin-labeled DNA probes complementary to the transcripts of Acps 29AB, 31F, 33A, 36DE, 53Ea, 63F, 76A or 98AB were hybridized in situ to RNA in whole mount accessory glands. In all cases, the probes hybridized strongly to mRNA in main cells (e.g. FIG. 2B for Acp36DE). In most cases the strong staining of main cells made it impossible to determine unambiguously whether there was also staining in the secondary cells. In the case of Acp36DE an accessory gland fortuitously positioned so that secondary cells were visible at the edge of the lobe was observed. In that case, it was possible to determine that the secondary cells were unstained (FIG. 2B, inset). Gene expression in main cells is stimulated in response to mating (Schmidt, 1985; DiBenedetto, 1990; Monsma, 1990; Bertram, 1992; Simmerl, 1995). This stimulation is also observed for the one new gene tested, Acp36DE (Bertram, 1994).

Example 4 General Features of the Predicted New Acps

The goal in isolating new Acp genes was to identify new molecules that could ultimately be correlated with the functions of the accessory gland. Therefore, the sequence of the predicted proteins encoded by 9 of the new genes was determined.

Of the two Acp genes with large transcripts, the one with the simpler transcription pattern, Acp36DE, was selected for sequence analysis. cDNAs were sequenced from all the smaller Acp transcripts for which cDNA clones were cloned (Acps 29AB, 33A, 32CD, 53Ea, 62F, 63F, 76A, 98AB). FIG. 3 reports the open reading frame sequence of each Acp, with FIG. 1 giving some of the salient numerical characteristics of the transcript and its open reading frame. In all cases but two, only a single long open reading frame in the Acp gene's transcript, beginning at the most 5′ AUG of the mRNA, was found. The two exceptions were Acps 33A and 36DE. The former encodes two open reading frames of similar size and codon bias; it is not known which (or if both) is the bona fide Acp ORF. For Acp36DE, the first AUG initiates a short open reading frame of 18 amino acids upstream of a 716 amino acid open reading frame. Antibodies raised against the protein predicted by the long open reading frame confirm that it is the expressed Acp (Bertram, 1996). As shown in FIG. 1, 4 bases prior to the initiating AUGs of all the Acp genes listed showed only partial matches to consensus for translational initiation in Drosophila (C/A A A C/A; Cavener, 1987). No consensus in these bases was seen among Acp genes.

Several features are apparent in the new predicted Acps (FIG. 3). First, all except Acp36DE contain predicted secretion signals. A sequence that matches a consensus for signal sequence cleavage (von Heijne, 1983) is found at the positions indicated in FIG. 3. Consistent with this observation, all Acps tested (29AB, 32CD, 53Ea, 62F, 63F and 98AB; Y.O.L., U.T. and M.F.W. unpublished, and 76A; Coleman, 1995) are secreted and transferred to females during mating. Despite its lack of an apparent signal sequence, Acp36DE is also secreted and transferred to females (Bertram, 1996). The predicted stretch of hydrophobic residues at its N-terminus may function as a secretion signal.

The second feature of the predicted Acps is that many contain sequences that suggest they may be subject to post-translational modifications such as cleavage, amidation and glycosylation. Basic amino acids in contexts favorable for cleavage to liberate peptide hormones from a precursor molecule are found in Acps 29AB, 32CD, 36DE, 53Ea, and 76A. Park and Wolfner (1995) have shown that cleavage of Acp26Aa occurs at sites consistent with the use of basic amino acids in such contexts (Schwartz, 1986; Benoit, 1987; Nakayama, 1992). Acp32CD contains a potential amidation site (173GKK; Kreil, 1984; Bradbury and Smyth, 1987). Acps 29AB, 32CD, 36DE, 53Ea, 62F, 63F and 76A contain sites for potential glycosylation (Kornfeld and Kornfeld, 1985; Miletich and Broze, 1990). The case of Acp36DE is particularly remarkable: in addition to a site for N-linked glycosylation (Asn439-Pro-Ser) and in fact being N-glycosylated (Bertram, 1996), Acp36DE contains large amounts of serine and threonine, at which O-linked glycosylation could occur. These residues are mostly concentrated in four regions: amino acids 42-64, 92-118, 334-351 and 591-665 of this glutamine-rich protein. Acp36DE also contains, along with Acps 32CD and 62F, sequences matching consensus for glycosaminoglycan attachment sites (Hassell, 1986; Bourdon, 1987), suggesting that these proteins could become associated with, or part of, an extracellular matrix. Taken together, these features suggest that the new Acps are secreted molecules, and some have the potential to be cleaved to smaller peptides and/or be glycosylated. The Acps that do not contain cleavage sites (Acps 33A, 62F 63F, 98AB) are within the size range of known secreted accessory gland peptides (e.g. Chen, 1988; Monsma and Wolfner, 1988) and thus might be synthesized and secreted without further modification.

Example 5 Intriguing Sequence Similarities in Two Predicted Acps

The databases were searched for sequences that might have similarities to the 9 new predicted Acps. Two showed sequence similarity to proteins in the data bases (FIGS. 3, 4).

Acp76A: A search of protein databases reveals that the 11 amino acids starting at position 345 and ending at 355, FEVNRPFVFVI (SEQ ID NO:33), are a perfect match to the serpin signature sequence, [LIVMF]-X-[LIVMFA]-[DNQ]-[RKHQ]-[PS]-F-[LIVMFY]-2X-[LIVMF] (SEQ ID NO:34) (Henikoff and Henikoff, 1994). This amino acid sequence similarity places the Acp76A in the serpin (serine protease inhibitors) superfamily of proteins. Members of this superfamily include alpha-1-antitrypsin, angiotensinogen, ovalbumin, antiplasmin, placental plasminogen activator inhibitor, thyroxin binding protein, and heparin cofactor II (Carrell and Boswell, 1986; Kanost, 1989). Many members of the serpin superfamily are extracellular serine protease inhibitors, helping to control proteolytic events associated with a wide variety of regulated biochemical pathways (Carrell and Boswell, 1986; Gettins, 1992; Zou, 1994). Several mammalian proteins in the serpin superfamily play roles in blood coagulation. Others are involved in fibrinolysis, inflammation, and tumor suppression. In humans a serpin, Protein C Inhibitor (PCI), is present in semen and interacts with at least three different proteases: urokinase plasminogen activator, tissue-type plasminogen activator, and prostate specific antigen (Espana, 1993). The physiological role of these interactions in human semen is unknown.

Although it is not yet known whether Acp76A, with its perfect serpin signature, is an active protease inhibitor, there are indications that protease inhibitors could play a critical role in controlling Acp function in D. melanogaster. Regulated proteolysis is a hallmark of Acp26Aa's fate in mated female flies (Monsma, 1990; Park and Wolfner, 1995). Acp36DE is also converted to a smaller form upon transfer to females (Bertram, 1996). Acp26Aa remains intact in the male's reproductive tract. It undergoes ordered proteolysis in the genital tract of the mated female, due to a combination of seminal fluid components donated by the male as well as components donated by the female (Park and Wolfner, 1995). It is possible that the protease(s) that cleaves Acp26Aa is made by the male's accessory gland, but is kept in check in the gland by protease inhibitors such as, potentially, Acp76A or the protease inhibitor found in D. funebris accessory glands (Schmidt, 1989). Upon transfer to the female, the protease inhibitor(s) would be inactivated, allowing the protease(s) to become active and to cleave Acp26Aa.

A second possible role for a protease inhibitor derived from accessory glands is in the coagulation of semen after mating. In Drosophila, as in many other insects, a mating plug consisting of coagulated semen forms in the mated female's uterus. The plug is thought to facilitate the storage of sperm by the female (Lefevre and Jonsson, 1962; Fowler, 1973; Bertram, 1996). By analogy to the role of members of the serpin superfamily in the tightly regulated proteolytic cascades that cause blood clotting in mammals (Gettins, 1992; Espana, 1993; Potempa, 1994), a serpin, potentially Acp76A, could function to regulate proteolytic cleavage events crucial to the formation of the mating plug. Consistent with this suggestion, Acp76A is transferred to the female fly during mating, and the majority of the transferred Acp76A is found in the mating plug when it is expelled from the female between 2-4 hours after mating (Coleman, 1995).

Acp62F: Acp62F has a 28 amino acid region of sequence similarity with several small neurotoxins made by the Brazilian “armed” spider, Phoneutria nigriventer. Sequence similarity is highest to the 48 amino acid toxin PhTx2-6, one of several related neurotoxins in the PhTx2 fraction of the P. nigriventer venom (Rezende, 1991; Cordeiro, 1992; Cordeiro, 1995). Optimal alignment of the Acp62F and PhTx2-6 sequences (FIG. 4) shows a 51.7% identity, 82.7% similarity in a region of 28 amino acid residues (aas 46-73) of Acp62F to a 27 amino acid region of PhTx2-6. In this alignment, all six of Acp62F's cysteines in this region are perfectly aligned with 6 of the 10 cysteines in PhTx2-6 and the other PhTx2 toxins. Furthermore, a seventh cysteine at the N-terminus of Acp62F is only 3 residues away from a corresponding cysteine in PhTx2-6.

PhTx2 toxins inhibit the closure of voltage-gated sodium channels in frog muscles in vitro, thus prolonging action potentials and increasing membrane excitability (Araujo, 1993; Cordeiro, 1995). If the sequence similarity observed between Acp62F and PhTx2-6 reflects a functional similarity, it could suggest roles for Acp62F in neuromuscular events following mating. Such roles could include opening the entry to the sperm storage organs to facilitate efficient storage of transferred sperm or removal of previously stored sperm, delaying movement of the first egg through the oviduct to allow sperm time to be stored before being pushed out by the egg or, alternatively, facilitating rapid release of eggs following mating.

The toxin-similarity of Acp62F is also intriguing in light of the demonstrated toxicity of D. melanogaster seminal fluid to mated female flies (Chapman, 1995; Rice, 1996). Acp62F enters the hemolymph after its entry into the mated Drosophila female. Since injection of toxin PhTx2-6 into the hemolymph of house flies is lethal (Rezende, 1991; Cordeiro, 1995), it is plausible that hemolymph entry of Acp62F could contribute to the decreased lifespan of mated female D. melanogaster (Fowler and Partridge, 1989; Chapman, 1995). Toxicity of seminal fluid is suggested to be an unintended negative consequence of a seminal fluid component that has a reproductively advantageous function (Chapman, 1995; Keller, 1995; Chapman and Partridge, 1996). Future genetic analysis of Acp62F is needed to determine whether this protein plays such a Jekyll-and-Hyde role.

Example 6 Lethality of Acp62F Expressed from a Baculovirus Vector in Trichoplusia ni

The survival of 5^(th) instar Trichoplusia ni was monitored after injection with a baculovirus vector carrying the gene encoding Acp62F. The gene encoding Acp62F was cloned into the BacPac vector from Clontech. This vector allows for the introduction and expression of the cloned gene in insect cells. Trichoplusia ni larva were injected with the baculovirus after reaching the 5^(th) instar stage of development. Two control groups were maintained, uninfected larva and vector injected larva. Survival was monitored at 2, 2.5, and 3 days post injection. The results shown in FIG. 5 were normalized relative to the survival of the control larva. A significant decrease in larval survival was seen with all the larva infected with baculovirus vectors carrying the Acp62 gene.

Example 7 Lethality of Acp62F Protein in Trichoplusia ni

Acp62F was injected into larvae at the 3^(rd) instar stage of development. Survival was monitored over a 12.5 day period after injection. Larvae were injected with 115 nano-grams of Acp62F. Control larvae were injected with an equal molar amount of BSA or an equal volume of saline solution. The percentage of surviving caterpillars was determined daily. The results are shown in FIG. 6. The percentage of caterpillars which pupated is also reported in Table 1. The percentage of pupating animals is virtually identical to the survival of the larva after 12.5 days. The injection of Acp62F protein clearly has an effect on the survival of the caterpillars. However, it may not be as effective as the introduction of a baculovirus which can continually express the protein due the half-life of the protein. TABLE 1 Pupation of Trichoplusia ni After Injection With Acp62F Protein % Pupation Uninjected n = 24 95.8% Saline n = 38 94.9% BSA n = 56 98.2% 62FH6 n = 55 78.2%

Example 8 Expression of Acp62F is Lethal to Pre-Adult Fruit Flies

The effect of accessory gland proteins on survival of pre-adult fruit flies was determined by inducing the expression of an accessory gland protein at different stages in the development of fruit fly larva. A fruit fly which is heterozygous for an accessory gland protein gene under the control of a heat inducible promoter was bred with a wild-type fly. The resulting progeny were expected to be 50% wild-type and 50% carriers of the heat inducible accessory gland protein gene. This allowed for cultures of flies with otherwise identical genetic makeup and at the identical developmental stage. Cultures were heat shocked on a particular day of development and then the flies were allowed to grow up (except for the group identified as “all,” which was heat shocked every day through the first 9 days of development). Normally 80-90% of eggs produced flies. A similar ratio of eggs developed into flies for those flies which were produced from flies carrying the Acp 26Aa, 29AB, 32CD, 33A, 53E, 63F/64A, and 92EF accessory gland protein genes (See FIG. 7).

On the other hand, only half as many flies resulted from eggs produced from a fly carrying an inducible Acp62F gene. Furthermore, when the flies were examined it was determined that the homozygous wild-type flies survived at the expected rates, but that few or no flies expressing the Acp62F gene had survived.

Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be made without departing from the spirit of the invention and these are therefore considered to be within the scope of the invention as defined in the claims which follow.

REFERENCES

The following references, which have been cited above, are hereby incorporated by reference:

Araujo, D. A. M., Cordeiro, M. N., C. R., D. and Beirao, P. S. L. (1993) Effects of a toxic fraction, PhTx2, from the spider Phoneutria nigriventer on the sodium current. Arch. Pharmacol. 347, 205-208.

Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A. and Struhl, K. (1994) Current Protocols in Molecular Biology. John Wiley and Sons. Inc.

Benoit, R., Ling, N. and Esch, T. (1987) A new prosomatostatin-derived peptide reveals a pattern for prohormone cleavage at monobasic sites. Science. 238, 1126-1129.

Bertram, M., Akerkar, G. A., Ard, R. L., Gonzalez, C. and Wolfner, M. F. (1992) Cell type specific gene expression in the Drosophila melanogaster male accessory gland. Mechs. Dev. 38, 33-40.

Bertram, M. J. (1994). Cell type-specific gene expression in the Drosophila melanogaster male accessory gland and the characterization of an accessory gland protein. Ph. D. thesis, Cornell University, Ithaca N.Y.

Bertram, M. J., Neubaum, D. M. and Wolfner, M. F. (1996) Localization of the Drosophila accessory gland protein Acp36DE in the mated female suggests a role in sperm storage. Ins. Bioch. Mol. Biol. 26, 971-980.

Bourdon, M. A., Krusius, T., Campbell, S., Schwartz, N. B. and Ruoslahti, E. (1987) Identification and synthesis of a recognition signal for the attachment of glycosaminoglycans to proteins. Proc. Natl. Acad. Sci. USA. 84, 3194-3198.

Bradbury, A. F. and Smyth, D. G. (1987) Biosynthesis of the C-terminal amide in peptide hormones. Biosci. Rep. 7, 907-916.

Bunge, J. and Fitzpatrick, M. (1993) Estimating the number of species: a review. J. Amer. Stats. Assoc. 88, 364-373.

Carrell, R. W. and Boswell, D. R. (1986). Serpins: a family of serine protease inhibitors. in Proteinase Inhibitors. New York, Elsevier. 403-420.

Cavener, D. R. (1987) Comparison of the consensus sequence flanking translational start sites in Drosophila and vertebrates. Nucl. Acids Res. 15, 1353-1361.

Chapman, T., Liddle, L. F., Kalb, J. M., Wolfner, M. F. and Partridge, L. (1995) Cost of mating in Drosophila melanogaster females is mediated by male accessory gland products. Nature. 373, 241-244.

Chapman, T. and Partridge, L. (1996) Sexual conflict as fuel for evolution. Nature. 381, 189-190.

Chen, P. S. (1991) Biochemistry and molecular regulation of the male accessory gland secretions in Drosophila (Diptera). Annls. Soc. ent. Fr. 27, 231-244.

Chen, P. S. (1996) The accessory gland proteins in male Drosophila: structural, reproductive and evolutionary aspects. Experientia. 52, 503-510.

Chen, P. S., Stumm-Zollinger, E., Aigaki, T., Balmer, J., Bienz, M. and Bohlen, P. (1988) A male accessory gland peptide that regulates reproductive behavior of female D. melanogaster. Cell. 54, 291-298.

Coleman, S., Drahn, B., Petersen, G., Stolorov, J. and Kraus, K. (1995) A Drosophila male accessory gland protein that is a member of the serpin superfamily of proteinase inhibitors is transferred to females during mating. Ins. Bioch. Mol. Biol. 25, 203-207.

Cordeiro, M., Diniz, C. R., Valentim, A. and Richardson, M. (1992) The purification and amino acid sequences of four Tx2 neurotoxins from the venom of the Brazilian “armed” spider Phoneutria nigriventer. FEBS Lett. 310, 153-156.

Cordeiro, M., Richardson, M., Gilroy, J., Gomes de Figuereido, S., Beirao, P. S. L. and Diniz, C. R. (1995) Properties of the venom from the South American “armed” spider Phoneutria nigriventer. J. Toxicol.-Toxin Rev. 14, 309-326.

Coulthart, M. B. and Singh, R. S. (1988) Differing amounts of genetic polymorphism in testes and male accessory glands of Drosophila melanogaster and Drosophila simulans. Bioch. Genet. 26, 153-164.

DiBenedetto, A. J., Harada, H. A. and Wolfner, M. F. (1990) Structure, cell-specific expression, and mating-induced regulation of a Drosophila melanogaster male accessory gland gene. Dev. Biol. 139, 134-148.

DiBenedetto, A. J., Lakich, D. M., Kruger, W. D., Belote, J. M., Baker, B. S. and Wolfner, M. F. (1987) Sequences expressed sex-specifically in Drosophila melanogaster adults. Dev. Biol. 119, 242-251.

Espana, F., Estelles, A., Fernandez, P. J., Giilabert, J., Sanchez-Cuenca, J. and Griffin, H. (1993) Evidence for the regulation of urokinase and tissue type plasminogen activators by the serpin, protein c inhibitor, in semen and blood plasma. Thromb. and Haem. 70, 989-994.

Esty, W. W. (1986) Estimation of the size of a coinage: a survey and comprison of methods. Numis. Chron. 146, 185-215.

Fowler, G. L. (1973) Some aspects of the reproductive biology of Drosophila: sperm transfer, sperm storage and sperm utilization. Adv. Genet. 17, 293-360.

Fowler, K. and Partridge, L. (1989) A cost of mating in female fruitflies. Nature. 338, 760-761.

Gettins, P., Patston, P. A. and Shapira, M. (1992) Structure and mechanism of action of serpins. Hemat./Onc. Clin. of N. Amer. 6, 1393-1408.

Goding, (1983) Monoclonal Antibodies: Principles and Practice, pp. 98-118, New York: Academic Press.

Goding, J., (1983) Monoclonal Antibodies: Principles and Practice, pp. 98-118 (N.Y. Academic press).

Good, I. J. (1950). Probability and the weighing of evidence. London, Charles Griffin.

Hall, J. C. (1994) The mating of a fly. Science. 264, 1702-1714.

E. Harlow, et. al., editors, Antibodies: A Laboratory Manual (1988).

Hassell, J. R., Kimura, J. H. and Hascall, V. C. (1986) Proteoglycan core protein families. Ann. Rev. Bioch. 55, 539-567.

Henikoff, S. and Henikoff, J. G. (1994) Protein family classification based on searching a database of blocks. Genomics. 19, 97-107.

Herndon, L. A. and Wolfner, M. F. (1995) A Drosophila seminal fluid protein, Acp26Aa, stimulates egg-laying in females for one day following mating. Proc. Natl. Acad. Sci. USA. 92, 10114-10118.

Ingman-Baker, J. and Candido, E. P. M. (1980) Proteins of the Drosophila melanogaster male reproductive system: two-dimensional gel patterns of proteins synthesized in XO. XY and XYY testis and paragonial gland and evidence that the Y chromosome does not code for structural sperm proteins. Biochem. Genet. 18, 809-828.

Kanost, M. R., Sarvamangala, V. P. and Wells, M. A. (1989) Primary structure of a member of the serpin superfamily of proteinase inhibitors from an insect, Manduca sexta. J. Biol. Chem. 264, 965.

Keller, L. (1995) All's fair when love is war. Nature. 373, 190-191.

Kelley, R. L. and Kuroda, M. I. (1995) Equality for X chromosomes. Science. 270, 1607-1610.

Kitts, P. A. and Possee, R. D. (1993) A Method for Producing Recombinant Baculovirus Expression Vectors at High Frequency. BioTechniques 14, 810-817

Kitts, P. A., Ayres, M. D., and Possee, R. D. (1990) Linearization of Baculovirus DNA Enhances the Recovery of Recombinant Virus Expression Vectors. Nuc. Acid. Res. 18, 5667-5672.

Kohler, G. and Milstein, C. (1975) Continuous Cultures of Fused Cells Secreting Antibody of Predefined Specificity. Nature 256, 495-497.

Kohler, G. and Milstein, C. (1976) Derivation of Specific Antibody-Producing Tissue Culture and Tumor Lines by Cell Fusion. Eur. J. Immunol. 6, 511-519.

Kornfeld, R. and Kornfeld, S. (1985) Assembly of asparagine-linked oligosaccharides. Ann. Rev. Biochem. 54, 631-664.

Kreil, G. (1984) Occurrence, detection and biosynthesis of carboxy-terminal amides. Meth. Enzymol. 106, 218-223.

Kubli, E. (1996) The Drosophila sex-peptide: a peptide pheromone involved in reproduction. Adv. Devel. Biochem. 4, 99-128.

Lehmann, R. and Tautz, D. (1994) In situ hybridization to RNA. in Meth. Cell Biol. 44: Drosophila melanogaster: practical uses in cell and molecular biology. L. S. B. Goldstein and E. A. Fyrberg, eds. Academic Press, San Diego Calif.. pp. 575-598.

Lefevre, G. and Jonsson, V. B. (1962) Sperm transfer, storage, displacement and utilization in Drosophila melanogaster. Genetics. 47, 1719-1736.

Luckow, V. A. and Summers, M. D. (1989) High Level Expression of Nonfused Foreign Genes with Autographa californica Nuclear Polyhedrosis Virus Expression Vectors. Virol. 17, 31-39.

Maniatis, T., Hardison, R. C., Lacy, E., Lauer, J., O'Connell, C., Quon, D., Sim, G. K. and Efstradiatis, A. (1978) The isolation of structural genes from libraries of eukaryotic DNA. Cell. 15, 687-701.

Miletich, J. P. and Broze, G. J. J. (1990) Beta protein C is not glycosylated at asparagine 329; the rate of translation may influence the usage at asparagine-X-cysteine sites. J. Biol. Chem. 265, 11397-11404.

Monsma, S. A., Harada, H. A. and Wolfner, M. F. (1990) Synthesis of two Drosophila male accessory gland proteins and their fate after transfer to the female during mating. Dev. Biol. 142, 465-475.

Monsma, S. A. and Wolfner, M. F. (1988) Structure and expression of a Drosophila male accessory gland gene whose product resembles a peptide pheromone precursor. Genes Dev. 2, 1063-1073.

Nakayama, K., Watanabe, T., Nakagawa, T., Kim, W. S., Nagahama, M., Hosaka, M., Hatsuzawa, K., Kondoh-Hashiba, K. and Murakami, K. (1992) Consensus sequence for precursor processing of mono-arginyl sites: evidence for the involvement of a kex2-like endoprotease in precursor cleavages at both dibasic and mono-arginyl sites. J. Biol. Chem. 267, 16335-16340.

Ohashi, Y. Y., Hamo-Fukushima, K. and Fuyama, Y. (1991) Purification and characterization of an ovulation stimulating substance from the male accessory glands of Drosophila suzukii. Insect Biochem. 21, 413-419.

Park, M. and Wolfner, M. F. (1995) Male and female cooperate in the processing of a Drosophila melanogaster seminal fluid protein. Dev. Biol. 171, 694-702.

Poole, S. J., Kauvar, L. M., Drees, B. and Kornberg, T. (1985) The engrailed locus of Drosophila: Structure analysis of an embryonic transcript. Cell. 40, 37-43.

Potempa, J., Korzus, E. and Travis, J. (1994) The serpin superfamily of proteinase inhibitors: Structure, function, and regulation. J. Biol. Chem. 269, 15957-15960.

Radka, S. F., Kostyu, D. D. and Amos, D. B. (1982) A Monoclonal Antibody Directed Against the HLA-Bw6 Epitope. J. Immunol. 128, 2804-2806.

Radka, S. F., Amos, D. B., Quackenbush, L. J., and Cresswell, P. (1984) HLA-DR7-Specific Monoclonal Antibodies and a Chimpanzee Anti-DR7 Serum Detect Different Epitopes on the Same Molecule. Immunogenetics 19, 63-76.

Rezende, L. J., Cordeiro, M. N., Oliveira, E. B. and Diniz, C. R. (1991) Isolation of neurotoxic peptides from the venom of the “armed” spider, Phoneutria nigriventer. Toxicon. 29, 1225-1233.

Rice, W. R. (1996) Sexually antagonistic male adaptation triggered by experimental arrest of female evolution. Nature. 381, 232-234.

Roberts, T. M. and Lauer, G. D. (1979) Maximizing Gene Expression on a Plasmid Using Recombination in Vitro. Methods in Enzymology, 68, 473-482.

Sambrook, (1989) Molecular Cloning: A Laboratory Manual, Cold Springs Laboratory, Cold Springs Harbor, N.Y.

Sanger, F., Nicklen, S. and Coulson, A. R. (1977) DNA sequencing with chain terminating inhibitors. Proc. Natl. Acad. Sci. USA. 74, 5463-5467.

Schäfer, U. (1986) Genes for male-specific transcripts in Drosophila melanogaster. Mol. Gen. Genet. 202, 219-225.

Schmidt, T., Choffat, Y., Schneider, M., Hunziker, P., Fuyama, Y. and Kubli, E. (1993) Drosophila suzukii contains a peptide homologous to the Drosophila melanogaster sex peptide and functional in both species. Ins. Bioch. Mol. Biol. 23, 571-579.

Schmidt, T., Stumm-Zollinger, E. and Chen, P. S. (1985) Protein metabolism of Drosophila melanogaster male accessory glands—III. Stimulation of protein synthesis following copulation. Insect Biochem. 15, 391-401.

Schmidt, T., Stumm-Zollinger, E. and Chen, P. S. (1989) A male accessory gland peptide with protease inhibitory activity in Drosophila funebris. J. Biol. Chem. 264, 9745-9749.

Schwartz, T. W. (1986) The processing of peptide precursors: proline-directed arginyl cleavage and other monobasic processing mechanisms. FEBS Lett. 200, 1-10.

Simmerl, E., Schäfer, M. and Schäfer, U. (1995) Structure and regulation of a gene cluster for male accessory gland transcripts in Drosophila melanogaster. Ins. Biochem. Molec. Biol. 25, 127-137.

Smith, G. E., Summers, M. D., and Fraser, M. J. (1983) Production of Human Beta Interferon in Insect Cells Infected with a Baculovirus Expression Vector. Mol. Cell. Biol. 3, 2156-2165.

“Stratagene Cloning Systems” Catalog (1993) from Stratagene, La Jolla, Calif.

Studier, F. W. et. al., (1990) “Use of T7 RNA Polymerase to Direct Expression of Cloned Genes,” Gene Expression Technology vol. 185.

Stumm-Zollinger, E. and Chen, P. S. (1985) Protein metabolism of Drosophila male accessory glands: I. Characterization of secretory proteins. Ins. Bioch. 15, 375-383.

Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987).

Tautz, D. and Pfeifle, C. (1989) A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback. Chromosoma. 98, 81-85.

von Heijne, G. (1983) Patterns of amino acids near signal sequence cleavage sites. Eur. J. Biochem. 135, 17-21.

Whalen, M. and Wilson, T. G. (1986) Variation and genomic localization of genes encoding Drosophila melanogaster male accessory gland proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Genetics. 114, 77-92.

Wolfner, M. F. (1997) Tokens of love: functions and regulation of Drosophila male accessory gland products. Ins. Bioch. Mol. Biol. 27, 179-192.

Zou, Z., Anisowicz, A., Hendrix, M. J. C., Thor, A., Neveu, M., Sheng, S., Rafidi, K., Seftor, E. and Sager (1994) Maspin, a serpin with tumor-suppressing activity in human mammary epithelial cells. Science. 263, 526-529. 

1. An isolated nucleic acid molecule encoding an accessory gland protein from Drosophila which has the biological property of a serine protease inhibitor.
 2. The isolated nucleic acid moleucle according to claim 1, wherein the nucleic acid molecule is selected from the group consisting of: a nucleic acid molecule having a nucleotide sequence according to SEQ ID NO:28; or a nucleic acid molecule which hybridizes to a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO:28 under hybridization conditions comprising hybridization in 0.9M SSC buffer at a temperature of 37° C. and washing with the SSC buffer at 37° C.
 3. The isolated nucleic acid molecule according to claim 1, wherein the accessory gland protein comprises an amino acid sequence according to SEQ ID NO:29.
 4. The isolated nucleic acid molecule according to claim 1, wherein the nucleic acid molecule is a deoxyribonucleic acid molecule.
 5. An expression vector comprising the nucleic acid molecule according to claim
 1. 6. The expression vector according to claim 5, wherein the expression vector is a viral vector.
 7. The expression vector according to claim 6, wherein the viral vector is a baculovirus vector.
 8. A host cell comprising the expression vector according to claim
 5. 9. The host cell according to claim 8, wherein the host cell is an insect cell.
 10. An isolated protein encoded by the nucleic acid molecule according to claim
 1. 11. An isolated Drosophila melanogaster accessory gland protein that has the biological property of a serine protease inhibitor, wherein said protein is selected from the group consisting of: a protein having an amino acid sequence according to SEQ ID NO:29; and a protein encoded by a nucleic acid molecule which hybridizes to a nucleic acid sequence according to SEQ ID NO:28 under hybridization conditions comprising hybridization in 0.9M SSC buffer at a temperature of 37° C. and washing with the SSC buffer at 37° C.
 12. The isolated Drosophila melanogaster accessory gland protein according to claim 11, wherein the accessory gland protein comprises an amino acid sequence according to SEQ ID NO:29.
 13. An isolated antibody, fragment thereof, or probe which recognizes a protein according to claim
 11. 14. An isolated antibody, fragment thereof, or probe according to claim 13, wherein the isolated antibody, fragment thereof, or probe is a monoclonal antibody.
 15. An isolated antibody, fragment thereof, or probe according to claim 13 wherein the isolated antibody, fragment thereof, or probe is a polyclonal antibody.
 16. A method for determining whether a female Drosophila melanogaster has recently mated comprising: providing an antibody, fragment thereof, or probe according to claim 13, wherein the antibody, fragment thereof, or probe is bound to a label effective to permit detection of said protein upon binding of the antibody, fragment thereof, or probe to said protein; contacting a fluid or tissue sample from the female Drosophila melanogaster with the antibody, fragment thereof, or probe having a label under conditions effective to permit binding of the antibody, fragment thereof, or probe to the protein; and detecting a presence of any of said protein in the biological sample by detecting the label. 